記述言語：英語   掲載種別：研究論文（学術雑誌）  

Platelet polyphosphate (polyP) can be conveniently quantified by exploiting a recent methodological breakthrough using 4',6-diamidino-2-phenylindole (DAPI). However, the preservation of these biological samples has not yet been standardized. In a preliminary study, potential protocols were screened, while accepted protocols were further tested in this study. Pure-platelet-rich plasma (P-PRP) samples and washed platelet suspensions were prepared using blood obtained from non-smoking healthy male donors and were fixed with ThromboFix for 20-24 h at 4 °C. Mass polyP levels were determined using a fluorometer at wavelengths of 425 and 525 nm. Platelet polyP levels were normalized to platelet counts. Statistical analyses were performed using non-parametric tests. Platelet polyP levels significantly decreased by 20% after 7 days in the platelet suspension maintained under fixed conditions at 4 °C (control). In contrast, the platelet polyP levels in both the P-PRP and washed platelet suspensions were maintained without a significant reduction for up to 6 weeks by removing ThromboFix after fixation and subsequent freezing in pure water at -80 °C. Fluorometric polyP quantification often interferes with the low specificity of DAPI binding and the wavelength used. Our validated protocols will enable long-term preservation and high-throughput polyP quantification and can be applied to relatively large cohort studies.

DOI： 10.3390/mps6040059

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Autologous platelet-rich plasma (PRP) therapy is ambiguously thought to be more effective in elite athletes than in sedentary patients, although the possible importance of recipient responsiveness remains poorly understood. To address this issue, along with the well-known PRP quality, in this initial study, we evaluated two candidate biomarkers: body composition indices (BCIs), which reflect systemic physical conditions, and resting platelet ATP levels, which reflect platelet energy expenditure and the mass of energy generation units. Methods: In this cross-sectional cohort study, blood samples were collected from male professional soccer players (PSPs) on a local professional team during the off-season and platelet ATP levels were quantified using an ATP luminescence assay kit. BCIs were measured using the body mass impedance method. Age-matched male sedentary participants were used as the controls. Results: Among the BCIs, the body mass index, basal metabolic rate (BMR), and skeletal muscle weight levels were higher in the PSPs than in the controls. The platelet ATP levels in the PSPs group were significantly lower than those in the control group. The correlation between BMR and platelet ATP levels was moderately negative in the control group, but weakly positive in the PSPs group. Conclusion: Owing to regular physical exercise, PSPs had higher BMR levels and lower platelet ATP levels without a significant mutual correlation compared to sedentary controls. This study did not indicate the influence of these biomarkers on the success of PRP therapy but provided evidence for a better understanding of PRP therapy, particularly for elite athletes.

DOI： 10.3389/fbioe.2023.1255860

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/s40729-022-00424-4

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その他リンク： https://link.springer.com/article/10.1186/s40729-022-00424-4/fulltext.html

担当区分：責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/s40729-022-00419-1

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その他リンク： https://link.springer.com/article/10.1186/s40729-022-00419-1/fulltext.html

記述言語：英語  

BACKGROUND: Regenerative therapy using platelet-rich plasma (PRP), a rich source of growth factors, has become popular in orthopedic sports medicine. Elite athletes prefer PRP therapy for their injured muscles and tendons primarily to avoid the possible risks of surgical treatment. However, the clinical effectiveness of PRP therapy in elite athletes compared to that in non-athletes remains unknown. Therefore, to investigate the effectiveness of PRP therapy in professional athletes (pro-athletes), we focused on the quality of PRP preparations and compared the levels of bioactive molecules between pro-athletes and non-athletes. METHODS: PRP was prepared from healthy, non-smoking male professional soccer players (pro-athletes) (n = 22) and non-athletes (VEGF: n = 34, others: n = 38). The levels of TGFβ1, PDGF-BB, VEGF, and PF4 were determined using ELISA kits. Polyphosphate was probed with 4',6-diamidino-2-phenylindole and monitored using a fluorometer. The body composition of the donors was determined using a bathroom weighing scale. RESULTS: The levels of TGFβ1 and VEGF were significantly lower in pro-athletes than in non-athletes, whereas PF4 levels were significantly higher in pro-athletes. No significant difference was found in PDGF-BB levels between these groups. Biomolecule levels were not correlated with polyphosphate levels. CONCLUSION: TGFβ1, VEGF, and PDGF-BB levels in pro-athletes were not higher than those in non-athletes. These findings suggest that growth factor levels in PRP may not be a predominant determinant of the clinical effectiveness of PRP therapy in pro-athletes. Increased PF4 levels in pro-athletes suggest an immunological function of PRP that may positively influence tissue regeneration.

DOI： 10.1186/s13018-022-03362-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.

DOI： 10.3390/ijms231911293

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.14814/phy2.15409

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.14814/phy2.15409

掲載種別：研究論文（学術雑誌）  

DOI： 10.3892/br.2022.1504

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/09537104.2020.1856359

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms22147257

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s00784-021-03825-8

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その他リンク： https://link.springer.com/article/10.1007/s00784-021-03825-8/fulltext.html

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.

DOI： 10.3390/ijms22042169

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掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/app11041666

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担当区分：責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/ijms22031040

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

<title>Abstract</title><sec>
<title>Background</title>
Platelet-rich plasma (PRP) is often used to improve surface biocompatibility. We previously found that platelets rapidly adhere to plain commercially pure titanium (cp-Ti) plates in the absence, but not in the presence, of plasma proteins. To further expand on these findings, in the present study, we switched titanium plates from a plain surface to a rough surface that is blasted with calcium phosphate (CaP) powder and then examined platelet adhesion and activation.


</sec><sec>
<title>Methods</title>
Elemental distribution in CaP-blasted cp-Ti plates was analyzed using energy-dispersive X-ray spectroscopy. PRP samples prepared from anticoagulated blood samples of six healthy, non-smoking adult male donors were loaded on CaP-blasted cp-Ti plates for 1 h and fixed for examination of platelet morphology and visualization of PDGF-B and platelet surface markers (CD62P, CD63) using scanning electron microscopy and fluorescence microscopy. Plain SUS316L stainless steel plates used in injection needles were also examined for comparison.


</sec><sec>
<title>Results</title>
Significant amounts of calcium and phosphate were detected on the CaP-blasted cp-Ti surface. Platelets rapidly adhered to this surface, leading to higher activation. Platelets also adhered to the plain stainless surface; however, the levels of adhesion and activation were much lower than those observed on the CaP-blasted cp-Ti plate.


</sec><sec>
<title>Conclusions</title>
The CaP-blasted cp-Ti surface efficiently entraps and activates platelets. Biomolecules released from the activated platelets could be retained by the fibrin matrix on the surface to facilitate regeneration of the surrounding tissues. Thus, PRP immersion could not only eliminate surface air bubbles but also improve the biocompatibility of the implant surface.


</sec>

DOI： 10.1186/s40729-020-00270-2

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その他リンク： http://link.springer.com/article/10.1186/s40729-020-00270-2/fulltext.html

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

Platelet-rich fibrin (PRF) is a fibrin matrix enriched with platelets. The PRF matrix is thought to form a steep gradient of platelet density around the region corresponding to the buffy coat in anticoagulated blood samples. However, this phenomenon has not yet been proven. To visualize platelet distribution in PRF in a non-invasive manner, we utilized near-infrared (NIR) imaging technology. In this study, four types of PRF matrices, bio-PRF, advanced-PRF (A-PRF), leukocyte-rich PRF (L-PRF), and concentrated growth factors (CGF) were compared. Blood samples collected from healthy, non-smoking volunteers were immediately centrifuged using four different protocols in glass tubes. The fixed PRF matrices were sagittally divided into two equal parts, and subjected to modified immunohistochemical examination. After probing with NIR dye-conjugated secondary antibody, the CD41+ platelets were visualized using an NIR imager. In L-PRF and CGF, platelets were distributed mainly on and below the distal surface, while in bio-PRF and A-PRF, platelet distribution was widespread and homogenous. Among three regions of the PRF matrices (upper, middle, and lower), no significant differences were observed. These findings suggest that platelets aggregate on polymerizing fibrin fibers and float up as a PRF matrix into the plasma fraction, amending the current “gradient” theory of platelet distribution.

DOI： 10.3390/ijms21124426

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers Media SA  

DOI： 10.3389/fbioe.2020.00600

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

It is generally accepted that citrate or the A-form of acid-citrate-dextrose (ACD-A) are suitable for preparing platelet-rich plasma (PRP) for regenerative therapy. However, this is based on evidence from blood transfusions and not from regenerative medicine. Thus, we examined the effects of anticoagulants, such as ACD-A, ethylenediaminetetraacetic acid (EDTA), and heparin, on the regenerative quality of PRP to address this gap. The blood samples were collected in the presence of anticoagulants and were processed to prepare pure-PRP. Platelet size, activation status, and intra-platelet free Ca2+ concentration were determined while using a hematology analyzer and flow cytometer. Platelet-derived growth factor-BB (PDGF-BB) was quantified while using an ELISA. In pure-PRP samples, EDTA caused platelet swelling and activation, but yielded the highest number of platelets. Heparin aggregated platelets and disturbed the overall counting of blood cells. However, no significant differences in PDGF-BB levels were observed among the anticoagulants tested. Moreover, when considering the easy preparation of platelet suspensions, without the need for high-level pipetting skills, these findings suggest the comparable potency of EDTA-derived pure-PRP in tissue regeneration and support the use of EDTA in the preparation of pure-PRP. Further in vivo studies are required in animal models to exclude the possible negative effects of including EDTA in pure-PRP preparations.

DOI： 10.3390/biomedicines8030042

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Because of its simple operation, platelet-rich fibrin (PRF) is becoming more popular than the original form, platelet-rich plasma (PRP), in regenerative dentistry. PRF preparation requires plain glass blood-collection tubes, but not either anticoagulants or coagulation factors. However, such glass tubes designed for laboratory testing are no longer commercially available. Although several glass tubes specifically designed for PRF preparation are available, many clinicians prefer to obtain stably supplied substitutes, such as silica-coated plastic tubes produced by major medical device companies. The quality of PRF prepared by silica-coated tubes has not been assessed and we previously reported significant contamination of silica microparticles in the resulting PRF matrix and alerted clinicians against the use for PRF preparation. To further assess the biosafety of the silica microparticles, we presently examined their effects on human normal periosteal cells derived from alveolar bone. The periosteal cells were obtained from explant cultures of small periosteal tissues obtained from healthy donors. Silica microparticles were obtained from silica-coated tubes and added to cell cultures. Cellular responses were monitored using a tetrazolium assay, phase-contract inverted microscopy, an immunofluorescence method, and scanning electron microscopy. Silica microparticles adsorbed onto the cell surface with seemingly high affinity and induced apoptosis, resulting in significant reduction of cell proliferation and viability. These findings suggest that silica microparticles contained in plastic tubes for the purpose of blood coagulation are hazardous for various cell types around sites where silica-contaminated PRF matrices are implanted.

DOI： 10.1007/s10266-020-00486-z

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DOI： 10.3390/dj7040109

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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DOI： 10.1111/jicd.12458

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者,　責任著者   記述言語：日本語  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The accurate detection of lymph node metastases is essential for treatment success in early-stage malignant cancer. Sentinel lymph node (SLN) biopsy is the most effective procedure for detecting small or micrometastases that are undetectable by conventional imaging modalities. To demonstrate a new approach for developing a more efficient SLN biopsy procedure, we reported a two-stage imaging method combining lymphoscintigraphy and near-infrared (NIR) fluorescence imaging to depict metastatic cancer cells in SLNs in vivo. Furthermore, the theranostic potential of the combined procedure was examined by cell culture and xenograft mouse model. Anti-HER2 and anti-epidermal growth factor receptor (EGFR) affibody probes were used for NIR fluorescence imaging. Strong NIR fluorescence signal intensity of the anti-EGFR affibody probe was observed in SAS cells (EGFR positive). Radioactivity in the SLNs was clearly observed in the in vivo studies. High anti-EGFR affibody NIR fluorescence intensity was observed in the metastatic lymph nodes in mice. The addition of the IR700-conjugated anti-EGFR affibody to the culture medium decreased the proliferation of SAS cells. Decreased proliferation was shown in Ki-67 immunohistochemistry in xenograft tumors. Our data suggest that a two-stage combined imaging method using lymphoscintigraphy and affibody probes may offer the direct visualization of metastatic lymph nodes as an easily applied technique in SLN biopsy. Although further animal studies are required to assess the effect of treating lymphatic metastasis in this approach, our study results provide a foundation for the further development of this promising imaging and treatment strategy for earlier lymph node metastasis detection and treatment.

DOI： 10.3390/ijms20020427

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

We investigated the effects of targeted functionalized silica nanoparticles on the radiosensitivity of cancer cells. Better control of the local concentration of silica nanoparticles may facilitate their use as an adjuvant in conjunction with ionizing radiation to target cancer cells while preventing damage to normal cells. Hyperbranched polyamidoamine (PAMAM) was grafted onto the surface of amorphous silica nanoparticles to functionalize them. The PAMAM-coated silica nanoparticles (PCSNs) were then conjugated with fluorescent dyes. Anti-HER2 antibodies were covalently attached to the labeled PCSNs. The HER2-overexpressing SK-BR3 breast cancer cell line was incubated in medium containing the PCSN probes. After incubation
the cells were exposed to X-ray radiation. Cells were counted in all samples using cell proliferation assays
and apoptotic cells were detected. The cell survival results showed that the combination of the targeted PCSN probes and radiation reduced the survival rate of SK-BR3 cells to a greater extent than when either PCSN probes, PCSNs or radiation were applied individually. The results also showed an increase in apoptosis in the SK-BR3 cells that internalized the PCSN probes and were then irradiated. Based on these data, PCSN probes act as specific radiosensitizing agents for HER2-overexpressing cells.

DOI： 10.3390/ijms19030908

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers Media S.A.  

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

DOI： 10.3389/fbioe.2018.00004

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Hindawi Limited  

Platelet concentrates (PCs), represented by platelet-rich plasma (PRP), have been widely applied in the fields of regenerative and aesthetic therapies. PCs' mechanisms of action, however, are too complicated, and it is not easy to present the whole picture
besides, clinical outcomes are hardly reproducible in many cases. Therefore, several medically advanced countries seemingly intend to regulate PC therapies weakly or strictly because of the increasing popularity. Japan established laws and regulations for PC therapy in the "Act on the Safety of Regenerative Medicine" along with the "Pharmaceuticals, Medical Devices and Other Therapeutic Products Act" in 2014, which, to our knowledge, represent the strictest regulatory framework for production and therapeutic use of PCs in the world. According to these laws and regulations, PCs produced for topical use should be prepared as cell-based medicinal products, essentially as should stem cells, in accordance with their registered ("licensed" under actual conditions) standard operating procedures. Nonetheless, criteria for their quality are not standardized. In this review, we discuss the quality of PC preparations by focusing on the basic concept and regulatory framework of regenerative medicine in Japan. Within the new framework, PC therapy is regulated by a specific notification and registration system, as is stem cell therapy. In comparison with the latter, however, risk factors that hamper successful PC therapy are much fewer. Via appropriate evaluation of patients' conditions and whole-blood samples by simple and sensitive but not yet fully standardized assays, it is theoretically possible that PC quality will be controlled nearly completely. In addition to or instead of standardization of preparation protocols, standardization of preoperative examination of individual PC preparations is an urgent task for improving and guaranteeing the safety and efficacy of PC therapy.

DOI： 10.1155/2018/6389157

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

The platelet-rich fibrin-like matrix (PRFM) is usually prepared onsite and immediately used for regenerative therapy. Nonetheless, to meet the clinical necessity of preserving the PRFM without quality deterioration, we developed a method for preparation of PRFMs from short-term-stored whole blood (WB) samples. In this study, to evaluate the practical expiration date of storage, we extended the storage time of WB samples from 2 to 7 days and assessed the quality of the resulting PRFMs. WB samples collected with acid-citrate-dextrose were stored with gentle agitation at ambient temperature. To prepare PRFMs, the stored WB samples were mixed with CaCl2 in glass tubes and centrifuged. Fibrin fiber networks, CD41 and CD62P expression, and Platelet Derived Growth Factor-BB (PDGF-BB) levels were examined by scanning electron microscopy (SEM), flow cytometry, and an Enzyme-Linked ImmunoSorbent Assay (ELISA), respectively. Long-term storage had no significant effect on either blood cell counts or platelet functions tested. The resulting PRFMs were visually identical to freshly prepared ones. PDGF-BB levels did not markedly decrease in a time-dependent manner. However, fibrin fibers gradually became thinner after storage. Although the coagulation activity may diminish, we propose that PRFMs can be prepared-without evident loss of quality-from WB samples stored for up to 7 days by our previously developed method.

DOI： 10.3390/biomedicines5030057

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：特定非営利活動法人 日本歯周病学会  

DOI： 10.2329/perio.59.68

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley-Blackwell  

A human-cultured alveolar bone-derived periosteal (hCP) sheet is an osteogenic grafting material used clinically in periodontal regenerative therapy, while platelet-rich fibrin (PRF), a platelet concentrate with fibrin clot, is considered to augment the wound healing process. Therefore, whether the combined use of hCP-PRF complex could facilitate bone regeneration synergistically was evaluated in animal models. Human periosteal segments (1 × 1 mm) were cultured initially on plastic dishes and formed an hCP sheet. The hCP sheet was implanted with freshly prepared human PRF into subcutaneous tissue (hCP: n = 4, hCP + PRF: n = 4) and 4 mm diameter calvarial bone defect models (hCP: n = 4, hCP + PRF: n = 4, control [defect-only]: n = 4) that prepared in nude mice. At 4 weeks postimplantation, new bone formation was evaluated by using μCT. Cell growth and neovascularization were evaluated by histochemical and immunohistological methods. In the subcutaneous tissue, mineral deposit formation, collagen deposition, and number of vessels were higher in the hCP + PRF group than in the hCP alone group. In the calvarial defect models, new bone formation was significantly higher in the hCP + PRF group than in the hCP alone group and defect-only control group. The numbers of vessels and PCNA-positive cells in calvarial defects were also increased in the hCP + PRF group more than in the hCP alone group. Platelet-rich fibrin preparations support the proliferation and the growth of periosteal cells to form well-combined active biological materials. Platelet-rich fibrin also stimulates the local angiogenesis in the implantation site. Therefore, the combined use of hCP and PRF could be clinically applicable in bone regeneration therapy.

DOI： 10.1002/cre2.71

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：日本膜学会  

Here, we prepared a poly(L–lactic acid) (PLLA) microporous membrane with open finger–like pores as a scaffold for tissue engineering by a nonsolvent–induced phase separation method with the aid of a surfactant. The increase of surfactant content in the polymer solution caused the mold (glass plate) side of the membrane to adopt an indented structure with open finger–like pores to enhance the internal surface area. Osteoblast–like cells inoculated on the indented side grew on and in the PLLA membrane scaffold to reach 2 ～ 3 times higher cell density per unit apparent area compared to that attained in monolayer cultures. The cells on the membrane deposited calcium compounds by osteoinduction with ascorbic acid 2–phosphate, dexamethasone, and β– glycerophosphate. The PLLA membrane with open finger–like pores will likely be useful as scaffolds to support the implantation of osteogenic cells in bone tissue engineering.

DOI： 10.5360/membrane.41.304

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その他リンク： http://search.jamas.or.jp/link/ui/2017289540

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Background and Purpose: Sinus lift (SL) using cultured autogenous periosteal cells (CAPCs) combined with autogenous bone and platelet-rich plasma (PRP) was performed to evaluate the effect of cell administration on bone regeneration, by using high-resolution three-dimensional computed tomography (CT).
Materials and Methods: SL with autogenous bone and PRP plus CAPC [CAPC(+)SL] was performed in 23 patients. A piece of periosteum taken from the mandible was cultured in M199 medium with 10% fetal bovine serum (FBS) for 6 weeks. As control, 16 patients received SL with autogenous bone and PRP [CAPC(-)SL]. Three-dimensional CT imaging was performed before and 4 months and 1 year after SL, and stratification was performed based on CT numbers (HUs) corresponding to soft tissue and cancellous or cortical bone.
Results: The augmented bone in CAPC(+)SL revealed an increase in HUs corresponding to cancellous bone as well as a decrease in HUs corresponding to grafted cortical bone. In addition, HUs corresponding to cancellous bone in the graft bed were increased in CAPC(+)SL but were decreased in CAPC(-)SL. Insertion torque during implant placement was significantly higher in CAPC(+)SL.
Conclusion: By promoting bone anabolic activity both in augmented bone and graft bed, CAPCs are expected to aid primary fixation and osseointegration of implants in clinical applications.

DOI： 10.1111/cid.12356

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MDPI AG  

We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m (Tc-99m) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with Tc-99m and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner.

DOI： 10.3390/ijms17071086

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MARY ANN LIEBERT, INC  

In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that gamma-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that gamma-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated gamma-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy.

DOI： 10.1089/bio.2015.0072

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/09553002.2016.1230242

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley-Blackwell  

Platelet-rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers (N = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84-fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79- and 5.51-fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose-dependent stimulation of periosteal cell proliferation in vitro.

DOI： 10.1002/cre2.26

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：John Wiley and Sons Inc.  

Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites
therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment.

DOI： 10.1002/jbm.b.33262

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Over the past decade, platelet-rich plasma (PRP), a platelet-concentrated plasma fraction, has been widely investigated and applied to regenerative medicine. The clinical utility of PRP is supported by evidence that PRP contains high concentrations of platelet-related growth factors and normal concentrations of plasma-derived fibrinogen, both of which contribute synergistically to the regenerative process. Additionally, its superior cost-efficacy versus conventional therapies is attractive to many clinicians. However, current disadvantages of PRP include a relatively complicated preparation procedure and variable operator-dependent efficacy. An additional disadvantage is the use of bovine thrombin, an animal-derived biological, as a coagulant. Many of these disadvantages are overcome by recent advances in preparation procedures and devices; for example, Joseph Choukroun simplified the platelet-rich fibrin preparation procedure and improved handling efficiency without the aid of animal-derived factors. With advancements in cell processing technology, there has been a general shift in cell therapy from autologous to allogeneic treatment; however, autologous PRP therapy will not easily be replaced by allogeneic treatment in the near future. Therefore, to provide more predictable regenerative therapy outcomes using autologous PRP, further investigations should address developing a standardized procedure for PRP preparation to augment its efficacy and potency, independent of donor variability. We would then propose that operators and clinicians prepare PRP according to the standardized protocol and to carefully evaluate the clinical scenario (i.e., recipient factors comprising skeletal defects) to determine which factor(s) should be added to PRP preparations. This careful approach will lead to improved clinical outcomes for patients.

DOI： 10.1007/s10266-015-0209-2

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Microfiltration membranes of poly(L-lactic acid) (PLEA) have been prepared by a nonsolvent-induced phase separation method with the aid of surfactants. Surfactants with hydrophilic-lipophilic balance (HLB) values of 14.9-15.6 were found to be useful in reducing the shrinkage in thickness of the PLEA membrane. Among the surfactants examined, Tween 80 was the best for preparing microfiltration membranes. The surfactant allowed instantaneous phase separation and seemed to enhance the diffusion of water in the PLEA solution during structure formation. The membrane had asymmetric finger-like structures and showed low membrane resistance and high bacterial cell retention when the membrane was prepared from a 10 wt% PLEA solution in 1,4-dioxane containing 10 wt.% Tween 80. Bovine serum albumin molecules passed through the membrane suggesting that the membrane functions as a microfiltration membrane. The membrane was stable at 25 degrees C but degradable at 60 degrees C in wet conditions. The membrane can be applied as a compostable microfiltration membrane in food and biochemical industries. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.memsci.2015.01.021

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jcyt.2014.08.005

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Mary Ann Liebert Inc.  

Radiation therapy for head and neck cancers often causes xerostomia (dry mouth) by acutely damaging the salivary glands through the induction of severe acute inflammation. By contrast, the mechanism underlying the X-ray-induced delayed salivary dysfunction is unknown and has attracted increasing attention. To identify and develop a mouse model that distinguishes the delayed from the acute effects, we examined three different mouse strains (C57BL/6, ICR, and ICR-nu/nu) that showed distinct T-cell activities to comparatively analyze their responses to X-ray irradiation. Three strains were irradiated with X-rays (25 Gy), and functional changes of the submandibular glands were examined by determining pilocarpine-induced saliva secretion. Structural changes were evaluated using histopathological and immunohistochemical examinations of CD3, cleaved poly (ADP-ribose) polymerase (PARP), and Bcl-xL. In C57BL/6 mice, the X-ray irradiation induced acute inflammation accompanied by severe inflammatory cell infiltration at 4 days postirradiation, causing substantial destruction and significant dysfunction at 2 weeks. Fibrotic repair was observed at 16 weeks. In ICR-nu/nu mice, the inflammation and organ destruction were much milder than in the other mice strains, but increased apoptotic cells and a significant reduction in salivary secretion were observed at 4 and 8 weeks and beyond, respectively. These results suggest that in C57BL/6 mice, X-ray-induced functional and structural damage to the salivary glands is caused mainly by acute inflammation. By contrast, although neither acute inflammation nor organ destruction was observed in ICR-nu/nu mice, apoptotic cell death preceded the dysfunction in salivary secretion in the later phase. These data suggest that the X-ray-irradiated ICR-nu/nu mouse may be a useful animal model for developing more specific therapeutic methods for the delayed dysfunction of salivary glands.

DOI： 10.1089/biores.2015.0017

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：John Wiley and Sons Inc.  

Platelet-rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previous in vitro and in vivo studies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP-induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time-lapse quantitative imaging demonstrated that in the initial phase (~2 h) of treatment, PRP substantially stimulated cell migration in a wound-healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45±0.16 mm and 0.82±0.23 mm in control and PRP-stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti-VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up-regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose- and time-dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up-regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short-term activation should be considered when the controlled release system of PRP components is designed.

DOI： 10.1002/cm.21221

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記述言語：日本語   出版者・発行元：THE MEMBRANE SOCIETY OF JAPAN  

Platelet–rich fibrin (PRF) was developed as an advanced form of platelet–rich plasma (PRP) and it is widely used as a source of growth factors for tissue regeneration. From this bio material, our laboratory made two important modifications. One was a thin PRF membrane prepared by the use of a novel PRF compression device. This method was capable of minimizing the loss of platelets and bio active growth factors and thereby enabled the PRF membrane to more effectively stimulate cell proliferation and neovascularization. To further clinically enhance the modified PRF membrane, it was exposed to mild heat compression technique. The heat compressed membrane was found to exhibit a longer period, three to four weeks, of chemical/physical stability. However, non–treated PRF membrane was resorbed within two weeks or less at implantation sites and therefore could barely maintain sufficient stability over an adequate amount of time for periodontal regeneration (bone and connective tissue). Compared with the control non–heat exposed gauze–compressed PRF, the resulting heat modified PRF membrane exhibited greater plasmin-resistant and remained stable for a significantly longer time period both in vitro and in vivo. Although additional modification may be required to further improve its clinical applicability, we suggest that this new modified membrane would be a promising bio material for guided tissue regeneration (GTR) treatment with the added advantage over currently available GTR membranes of contributing growth factors to improve regeneration potential.

DOI： 10.5360/membrane.40.118

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その他リンク： https://jlc.jst.go.jp/DN/JLC/20011687830?from=CiNii

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

An atmospheric-pressure plasma (APP) treatment was recently reported to render titanium (Ti) surfaces more suitable for osteoblastic cell proliferation and osteogenesis. However, the mechanism of action remains to be clearly demonstrated. In this study, we focused on cell adhesion and examined the effects of the APP treatment on the initial responses of human prenatal-derived osteoblastic cells incubated on chemically polished commercially pure Ti (CP-cpTi) plates. In the medium containing 1% fetal bovine serum, the initial cell adhesion and the actin polymerization were evaluated by scanning electron microscopy and fluorescence microscopy. The expression of cell adhesion-related molecules and osteoblast markers at the messenger RNA level was assessed by real-time quantitative polymerase chain reaction. Although the cells on the APP-treated CP-cpTi surface developed fewer cytoskeletal actin fibers, they attached with higher affinity and consequently proliferated more actively (1.46-fold over control at 72 h). However, most of the cell adhesion molecule genes were significantly downregulated (from 40 to 85% of control) in the cells incubated on the APP-treated CP-cpTi surface at 24 h. Similarly, the osteoblast marker genes were significantly downregulated (from 49 to 63% of control) at 72 h. However, the osteoblast marker genes were drastically upregulated (from 197 to 296% of control) in these cells by dexamethasone and beta-glycerophosphate treatment. These findings suggest that the APP treatment improves the ability of the CP-cpTi surface to support osteoblastic proliferation by enhancing the initial cell adhesion and supports osteoblastic differentiation when immature osteoblasts begin the differentiation process. (C) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/jbm.b.33114

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Informa Healthcare  

Background aims: Cultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell-extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms ofperiosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, andthree-dimensional hybrid mock-ups of cells dispersed onto collagen sponges. Methods: Periosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively. Results: Real-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium-expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets. Conclusions: Integrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets. © 2014 International Society for Cellular Therapy.

DOI： 10.1016/j.jcyt.2013.11.003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Background: Molecular biomarkers are essential for monitoring treatment effects, predicting prognosis, and improving survival rate in oral squamous cell carcinoma. This study sought to verify the effectiveness of two integrin gene expression ratios as biomarkers.Methods: Gene expression analyses of integrin α3 (ITGA3), integrin β4 (ITGB4), CD9 antigen (CD9), and plakoglobin (JUP) by quantitative real-time PCR were conducted on total RNA from 270 OSCC cases. The logrank test, Cox proportional hazards model, and Kaplan-Meier estimates were performed on the gene expression ratios of ITGA3/CD9 and ITGB4/JUP and on the clinicopathological parameters for major clinical events.Results: A high rate (around 80%) of lymph node metastasis was found in cases with a high ITGA3/CD9 ratio (high-ITGA3/CD9) and invasive histopathology (YK4). Primary site recurrence (PSR) was associated with high-ITGA3/CD9, T3-4 (TNM class), and positive margin, indicating that PSR is synergistically influenced by treatment failure and biological malignancy. A high ITGB4/JUP ratio (high-ITGB4/JUP) was revealed to be a primary contributor to distant metastasis without the involvement of clinicopathological factors, suggesting intervention of a critical step dependent on the function of the integrin β4 subunit. Kaplan-Meier curves revealed positive margin as a lethal treatment consequence in high-ITGA3/CD9 and YK4 double-positive cases.Conclusion: Two types of metastatic trait were found in OSCC: locoregional dissemination, which was reflected by high-ITGA3/CD9, and distant metastasis through hematogenous dissemination, uniquely distinguished by high-ITGB4/JUP. The clinical significance of the integrin biomarkers implies that biological mechanisms such as cancer cell motility and anchorage-independent survival are vital for OSCC recurrence and metastasis. © 2013 Nagata et al.
licensee BioMed Central Ltd.

DOI： 10.1186/1471-2407-13-410

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously published an investigation indicating freeze-dried platelet-rich plasma (PRP)-coated polyglactin mesh was a promising wound-dressing material. However, one of its disadvantages was the inflammatory nature due to degradation of the polyglactin. Therefore, in this study, we investigated the use of a collagen sponge as the carrier for PRP. When implanted subcutaneously in nude mice, the PRP-coated sponge alone rapidly induced angiogenesis and infiltration of surrounding connective tissue without inducing appreciable inflammation. Moreover, addition of periosteal fibroblastic cells substantially augmented the angiogenic response. With in vitro studies, the PRP-coated sponge provided various major growth factors at high levels to stimulate the proliferation of cells cultured on plastic dishes, but did not stimulate the proliferation of cells inoculated into the PRP-coated sponge. Cells were embedded in the fibrin mesh and maintained their spherical shape without stretching. The atomic force microscopic analysis demonstrated that the fibrin gel formed on the PRP-coated sponge was much softer (approx. 22. kPa) than the cross-linked collagen that formed the sponge base (appox. 1.9. MPa). Because insoluble matrices have recently and increasingly been considered important regulatory factors of cellular behavior, as are soluble growth factors, it is suggested that this soft fibrin mesh possibly suppresses cell survival. Overall, our investigation has successfully demonstrated improved wound-healing and regenerative potential of the PRP-coated mesh by combining it with the collagen sponge. In the clinical setting, this PRP-coated collagen sponge is a promising material for connective tissue regenerative therapy, such as periodontal therapy, burn victim treatment and in cosmetic or plastic surgery. © 2013 Elsevier Inc.

DOI： 10.1016/j.cryobiol.2013.01.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66. GPa). ⋙. dispersed (9.99. kPa). &gt
. central region (5.20. kPa). &gt
. peripheral regions (3.67. kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed. &gt
. central region. &gt
. peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40. kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site. © 2013 Elsevier Ltd.

DOI： 10.1016/j.micron.2013.02.001

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記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：Nova Science Publishers, Inc.  

Mast cells are resident cells in a variety of organ tissues, and are known as a key cell type involved in type I hypersensitivity.However, recent studies suggest that these cells may be capable of regulating inflammation, host defense, and innate immunity.Moreover, these cells have been associated with the pathophysiology of inflammatory bowel disease (IBD), which consists of ulcerative colitis and Crohn's disease. IBD is characterized by chronically relapsing inflammation of the bowel of unknown origin. Markedly increased numbers of mast cells were observed in the mucosa of the ileum and colon of patients with IBD, which was accompanied by elevated degranulation of chemical mediators and cytokines such as histamine, tryptase, substance P and Tumor necrosis factor (TNF)-α. These data indicate the mast cell contribution to the pathophysiological process in IBD, suggesting that regulation of mast cells could be a therapeutic strategy for IBD. Regulation of mast cell activation by mast cell stabilizing agents could reduce inflammation and fibrosis of intestinal lesions of the experimental colitis.Additionally, it was shown that attenuation of experimental colitis was observed in mast cell-deficient mice as compared with wild type mice. In clinical settings, oral or rectal administration of tranilast, N-(3', 4'-dimethoxycinnamonyl)- anthranilic acid, a mast cell stabilizing agent, has been used empirically for patients with ulcerative colitis in Japan. Oral administration of tranilast also improved the stenotic lesions of small bowel of the patients with Crohn's disease.However, the therapeutic mechanisms of tranilast for the intestinal lesions of IBD are largely unknown.In this review, we discussed about the role of mast cells in IBD, especially their role in intestinal fibrosis of Crohn's disease.We proposed a hypothesis of a crosstalk between mast cells and mesenchymal cells, including fibroblasts, in the gut wall of patients with Crohn's disease.We suggest that mast cells could be potential therapeutic targets in intestinal inflammation and fibrosis of IBD. © 2013 Nova Science Publishers, Inc. All rights reserved.

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記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：Nova Science Publishers, Inc.  

Vimentin is the major structural component of intermediate filaments in cells of mesenchymal origin such as fibroblasts, and myofibroblasts, which maintain the extracellular matrix (ECM) of the fibrous connective tissue. In wound healing process, these vimentin-positive mesenchymal cells play an important role by producing ECM molecules and its over-production finally leads to tissue fibrosis. A frequent complication of Crohn's disease (CD), one of the two major forms of inflammatory bowel disease, is fibrosis leading finally to intestinal strictures. The profibrogenic mesenchymal cells such as fibroblasts, myofibroblasts, and smooth muscle cell (SMC)-like cells are supposed to produce ECM proteins, accumulating prominently in the submucosa of intestinal fibrotic lesions of CD. There is a controversy about the origin of the fibrogenic cells in stricture lesions in CD. Mechanistic studies of intestinal fibrosis are difficult to perform in humans, therefore, we investigated the lesions of chronic fibrosing colitis induced by dextran sulfate sodium (DSS), with special relevance to the role of vimentin-positive mesenchymal cells in the lesions. Our investigations revealed that fibroblasts (vimentin(V)+)/α SMA(A)-) were increased in both mucosal and submucosal layer, while myofibroblasts (V+/A+) were increased in mucosal but not submucosal layers. Interestingly, irsogladine maleate, a drug for anti-ulcer disease, ameliorated inflammation and fibrosis in this chronic fibrosing DSS colitis with decreased number of these vimentin-positive mesenchymal cells. In conclusion, vimentin-positive profibrogenic mesenchymal cells cause intestinal fibrosis in the chronic DSS colitis model. Therefore, we suggest that these cells could be promising therapeutic targets in intestinal fibrosis of CD. © 2013 Nova Science Publishers, Inc. All rights reserved.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.scr.2012.08.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Flexible microporous scaffold membranes of poly(l-lactic acid) (PLLA)/poly(ε-caprolactone) (PCL) blend were developed for bone regeneration. The new membranes overcome the fragility problems of PLLA membranes. When the PCL content in the blend was increased to 50%, the mechanical property for elongation was significantly improved without sacrificing the pores on the membrane surface that help tissue segments adhere the membrane. However, further increases in PCL content reduced the surface pores. In addition, spontaneous cell invasion and formation of cell-multilayers were observed in the membrane of PLLA/PCL blend (50:50). These results indicate that the porous membrane of PLLA/PCL blend (50:50) is useful for the preparation of periosteal sheets in tissue engineering. © 2012 Elsevier B.V.

DOI： 10.1016/j.matlet.2012.12.076

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

One-year data after autologous grafting of infrabony periodontal defects with human cultured periosteum sheets in combination with platelet-rich plasma and hydroxyapatite granules have shown favorable clinical and radiographic results. A 5-year follow-up evaluation of 22 selected patients indicated that treated infrabony defects remained stable. Radiographically, there was an increase in osseous radiopacity and bone trabeculation suggesting further bone maturation. This novel tissue-engineered periodontal treatment approach has resulted in significant clinical improvement, and defects remained stable after 5 years. © 2013 by Quintessence Publishing Co Inc.

DOI： 10.11607/prd.1545

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SAGE Publications Ltd  

As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum-supplemented STK1 for 28 days, in 1% human serum-supplemented STK1 for 14 days followed by 1% human serum-supplemented STK3 for 14 days (1% human serum-supplemented STK1+3), or in 10% fetal bovine serum-supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum-supplemented STK1 and 1% human serum-supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum-supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum-supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum-supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum-supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material. © The Author(s) 2013.

DOI： 10.1177/2041731413509646

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Owing to the necessity for the immediate preparation from patients' blood, autologous platelet-rich plasma (PRP) limits its clinical applicability. To address this concern and respond to emergency care and other unpredictable uses, we have developed a freeze-dried PRP in an adsorbed form on a biodegradable polymer material (Polyglactin 910). On the polymer filaments of PRP mesh, which was prepared by coating the polymer mesh with human fresh PRP and subsequent freeze-drying, platelets were incorporated, and related growth factors were preserved at high levels. This new PRP mesh preparation significantly and reproducibly stimulated the proliferation of human periodontal ligament cells in vitro and neovascularization in a chorioallantoic membrane assay. A full-thickness skin defect model in a diabetic mouse demonstrated the PRP mesh, although prepared from human blood, substantially facilitated angiogenesis, granulation tissue formation, and re-epithelialization without inducing severe inflammation in vivo. These data demonstrate that our new PRP mesh preparation functions as a bioactive material to facilitate tissue repair/regeneration. Therefore, we suggest that this bioactive material, composed of allogeneic PRP, could be clinically used as a promising alternative in emergency care or at times when autologous PRP is not prepared immediately before application. Copyright © 2012 Informa UK Ltd.

DOI： 10.3109/09537104.2011.645923

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Upon clinical application, thick platelet-rich fibrin (PRF) is usually compressed to fit the implantation site. However, it is speculated that the preservation of platelets and plasma content depends on the compression methods used. To accurately evaluate the clinical outcome of PRF, the preparation protocol should be standardized. Freshly prepared PRF clots were compressed into a thin membrane by our novel PRF compression device. The localization of platelets was examined by SEM and immunostaining. Growth factor levels were evaluated by bioassays and cytokine-antibody array techniques. The angiogenic activity was examined by the chick chorioallantoic membrane assay and the scratch assay using HUVEC cultures. Platelets were concentrated on the surface of the region adjacent to the red thrombus and this region was subjected to the experiments. Compared to the PRF membrane compressed by dry gauze (G-PRF), the preservation of the plasma content, 3D-fibrin meshwork, and platelets was more intact in the compressor-prepared PRF membrane (C-PRF). Among the growth factors tested, C-PRF contained PDGF isoforms at higher levels, and significantly stimulated cell proliferation and neovascularization. C-PRF may be useful for grafting while minimizing the loss of bioactive factors. This C-PRF preparation protocol is proposed as a standardized protocol for PRF membrane preparation. © 2012 The International Alliance for Biological Standardization.

DOI： 10.1016/j.biologicals.2012.07.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER JAPAN KK  

Intestinal fibrosis is a common and severe complication of inflammatory bowel disease (IBD), especially Crohn's disease (CD). To investigate the therapeutic approach to intestinal fibrosis, we have developed a mouse model of intestinal fibrosis by administering dextran sulfate sodium (DSS) and examining the effects of irsogladine maleate (IM) [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate], which has been widely used as an antiulcer drug for gastric mucosa in Japan, on DDS-induced chronic colitis. In this experimental colitis lesion, several pathognomonic changes were found: increased deposition of collagen, increased number of profibrogenic mesenchymal cells such as fibroblasts (vimentin(+), alpha-SMA(-)) and myofibroblasts (vimentin(+), alpha-SMA(+)) in both mucosa and submucosa of the colon with infiltrating inflammatory cells, and increased mRNA expressions of collagen type I, transforming growth factor (TGF)-beta, matrix metalloproteinase (MMP)-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-1. When IM was administered intrarectally to this colitis, all these pathological changes were significantly decreased or suppressed, suggesting a potential adjunctive therapy for intestinal fibrosis. IM could consequently reduce fibrosis in DSS colitis by direct or indirect effect on profibrogenic factors or fibroblasts. Therefore, the precise effect of IM on intestinal fibrosis should be investigated further.

DOI： 10.1007/s00795-011-0550-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1089/bio.2011.0051

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In ongoing clinical research into the use of cultured autogenous periosteal cells (CAPCs) in alveolar bone regeneration, CAPCs were grafted into 33 sites (15 for alveolar ridge augmentation and 18 for maxillary sinus lift) in 25 cases. CAPCs were cultured for 6. weeks, mixed with particulate autogenous bone and platelet-rich plasma, and then grafted into the sites. Clinical outcomes were determined from high-resolution three-dimensional computed tomography (3D-CT) images and histological findings. No serious adverse events were attributable to the use of grafted CAPCs. Bone regeneration was satisfactory even in cases of advanced atrophy of the alveolar process. Bone biopsy after bone grafting with CAPCs revealed prominent recruitment of osteoblasts and osteoclasts accompanied by angiogenesis around the regenerated bone. 3D-CT imaging suggested that remodeling of the grafted autogenous cortical bone particles was faster in bone grafting with CAPCs than in conventional bone grafting. The use of CAPCs offers cell-based bone regeneration therapy, affording complex bone regeneration across a wide area, and thus expanding the indications for dental implants. Also, it enables the content of particulate autogenous bone in the graft material to be reduced to as low as 40%, making the procedure less invasive, or enabling larger amounts of graft materials to be prepared. It may also be possible to dispense with the use of autogenous bone altogether in the future. The results suggest that CAPC grafting induces bone remodeling, thereby enhancing osseointegration and consequently reducing postoperative waiting time after dental implant placement. © 2012 Elsevier Inc.

DOI： 10.1016/j.bone.2012.02.631

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担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

In this in vitro study, novel porous poly(l-lactic acid) membranes were developed to improve periosteal sheets by promoting initial adhesion of periosteal tissue segments and stimulating the formation of a viable multilayered cellular sheet. The biocompatibility, biodegradability, and osteogenicity were evaluated using human periosteal tissue segments cultured on porous poly(l-lactic acid) membranes
the periosteal sheets were osteogen induced and were then implanted in the dorsal subcutaneous tissue of nude mice. In vivo, the membrane degraded into clusters of membrane particles separated by wide cracks
fibroblastic cells invaded along with small blood vessels from the surrounding mouse connective tissue. In osteoinduced periosteal sheets, the membrane clusters were surrounded by numerous capillaries and a number of tartrate-resistant acid phosphatase-positive, multinucleated cells. Neither severe inflammation nor fibrous encapsulation was observed throughout the implantation (∼12 weeks). These porous poly(l-lactic acid) membranes were highly biocompatible and functioned well as biodegradable scaffolds that could enhance the use of osteogenic periosteal sheets in therapy. © The Author(s) 2012.

DOI： 10.1177/0883911512438306

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC MEDICINE PRESS LTD  

Background: In a previous study using a rodent osteosarcoma-grafted rat model, in which cell-dependent mineralization was previously demonstrated to proportionally increase with growth, we performed a quantitative analysis of mineral deposit formation using (99m)Tc-HMDP and found some weaknesses, such as longer acquisition time and narrower dynamic ranges (i.e. images easily saturated). The recently developed near-infrared (NIR) optical imaging technique is expected to non-invasively evaluate changes in living small animals in a quantitative manner.
Purpose: To test the feasibility of NIR imaging with a dual-channel system as a better alternative for bone scintigraphy by quantitatively evaluating mineralization along with the growth of osteosarcoma lesions in a mouse-xenograft model.
Material and Methods: The gross volume and mineralization of osteosarcoma lesions were evaluated in living mice simultaneously with dual-channels by NIR dye-labeled probes, 2-deoxyglucose (DG) and pamidronate (OS), respectively. To verify these quantitative data, retrieved osteosarcoma lesions were then subjected to ex-vivo imaging, weighing under wet conditions, microfocus-computed tomography (mu CT) analysis, and histopathological examination.
Results: Because of less scattering and no anatomical overlapping, as generally shown, specific fluorescence signals targeted to the osteosarcoma lesions could be determined clearly by ex-vivo imaging. These data were well positively correlated with the in-vivo imaging data (r &gt; 0.8, P &lt; 0.02). Other good to excellent correlations (r &gt; 0.8, P &lt; 0.02) were observed between DG accumulation and tumor gross volume and between OS accumulation and mineralization volume.
Conclusion: This in-vivo NIR imaging technique using DG and OS is sensitive to the level to simultaneously detect and quantitatively evaluate the growth and mineralization occuring in this type of osteosarcoma lesions of living mice without either invasion or sacrifice. By possible mutual complementation, this dual imaging system might be useful for accurate diagnosis even in the presence of overlapping tissues.

DOI： 10.1258/ar.2011.110131

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Human cultured periosteal sheets, which are developed from small excised periosteum tissue segments (PTSs) in culture dishes by simple expansion culture, have been applied as a promising autologous osteogenic grafting material for periodontal regenerative therapy. However, the weak initial adhesion of PTSs to dish surfaces often hampers cellular outgrowth and limits the number of preparations. To correct this weakness and still avoid the use of animal-derived adhesion biomolecules, we have developed a novel, biodegradable, porous poly(L-lactic acid) (pPLLA) membrane. Freshly excised PTSs bound well to the highly porous pPLLA membrane, possibly due to the presence of semihemispheric 20-30 mu m diameter openings on the upper surface. Global gene expression analysis demonstrated that periosteal sheets cultured on pPLLA membranes upregulated expression of many adhesion molecules. Osteogenic induction stimulated the production of proteoglycans by these cells and concomitantly enhanced their expansion and penetration into the deep pore regions of the membrane in parallel with the progression of in vitro mineralization. These findings suggest that our pPLLA membranes not only facilitate initial adhesion, primarily mediated by adsorbed proteins, but also enhance biological adhesion by inducing endogenous adhesion molecules in periosteal sheet cultures. Therefore, the efficacy of periosteal sheets in therapy should be greatly enhanced by using this new pPLLA membrane. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 98A: 100-113, 2011.

DOI： 10.1002/jbm.a.33074

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER/PLENUM PUBLISHERS  

Microfocus X-ray computed tomography (mu CT) has now become widely available for the nondestructive evaluation of porous bioceramics suitable for use as a bone substitute in orthopedic surgery. As part of an official Japanese working committee, we recently participated in the preparation of a proposed standard protocol for the quantitative mu CT analysis of porous bioceramics sent to the International Organization for Standardization (ISO). In this protocol, the recommended basic conditions for analysis were [field of view (XY plane): 3.0 mm, spatial resolution: 6 mu m/pixel (or the closest minimal values available for both parameters on a particular mu CT system), matrix size: 512 pixels], and we have now further determined the optimal values for more detailed parameters (e.g., threshold determination). To validate the utility of the complete protocol, three different types of ceramic sample [a ceramic of beta-tricalcium phosphate (beta-TCP) and two types of hydroxyapatite (HAp) with different porosities] were evaluated with three different types of cone-beam mu CT scanner (the Shimadzu SMX-100CT, Shimadzu inspeXio-90CT, and Skyscan-1174 scanners). Acquired images were quantified using 3D-reconstruction software, VGStudio MAX (version 1.2). After comparing data obtained from these three mu CT scanners, we have found that determinations of both porosity and pore-interconnectivity were very similar from one system to the other although the total number of measured pores did vary between scanners. The present data indicate that our protocol for mu CT analysis is reliable enough to quantify the porosity and interconnectivity of porous bioceramics and would therefore facilitate both large-scale screening and quality control of porous bioceramic samples.

DOI： 10.1007/s10921-011-0092-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199 + 10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75 degrees C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37 degrees C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved FTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cryobiol.2011.03.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE INC  

Activation of osteoblastic bone anabolism in the calvarial sutures is considered to be the essential pathologic condition underlying mutant FGFR2-related craniofacial dysostosis. However, early clinical investigations indicated that abnormal cartilage development in the cranial base was rather a primary site of abnormal feature in Apert Syndrome (AS). To examine the significance of cartilaginous growth of the cranial base in AS, we generated a transgenic mouse bearing AS-type mutant Fgfr2IIIc under the control of the Col2a1 promoter-enhancer (Fgfr2IIIc(P253R) mouse). Despite the lacking expression of Fgfr2IIIc(P253R) in osteoblasts, exclusive disruption of chondrocytic differentiation and growth reproduced AS-like acrocephaly accompanied by short anterior cranial base with fusion of the cranial base synchondroses, maxillary hypoplasia and synostosis of the calvarial sutures with no significant abnormalities in the trunk and extremities. Gene expression analyses demonstrated upregulation of p21, Ihh and Mmp-13 accompanied by modest increase in expression of Sox9 and Runx2, indicating acceleration of chondrocytic maturation and hypertrophy in the cranial base of the Fgfr2IIIc(P253R) mice. Furthermore, an acquired affinity and specificity of mutant FGFR2IIIc(P253R) receptor with FGF2 and FGF10 is suggested as a mechanism of activation of FGFR2 signaling selectively in the cranial base. In this report, we strongly suggest that the acrocephalic feature of AS is not alone a result of the coronal suture synostosis, but is a result of the primary disturbance in growth of the cranial base with precocious endochondral ossification. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bone.2010.11.014

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Fibrogenic mesenchymal cells including fibroblasts and myofibroblasts play a key role in intestinal fibrosis, however, their precise role is largely unknown. To investigate their role in intestinal fibrosis, we analyzed the lesions of chronic colitis in C57BL/6 (B6) mice induced by dextran sulfate sodium (DSS). B6 mice exposed to single cycle administration of DSS for 5 days developed acute colitis that progressed to severe chronic inflammation with dense infiltrates of mononuclear cells, irregular epithelial structure, thickening of colonic wall, and persistent deposits of collagen. Increased mRNA expressions of proinflammatory cytokines are correlated with extensive cellular infiltration, and the mRNA expressions of collagen 1, transforming growth factor (TGF)-beta, and matrix metalloproteinases were also enhanced in the colon. In the colon of chronic DSS colitis, fibroblasts (vimentin+, alpha-smooth muscle actin (alpha-SMA)-) were increased in both mucosal and submucosal layers, while myofibroblasts (vimentin+, alpha-SMA+) were increased in mucosal but not in submucosal layers. Primary mouse subcutaneous fibroblast cultures experiments revealed that exogenously added TGF-beta 1 substantially augmented the expressions of both vimentin and alpha-SMA proteins with increased production of collagen. In conclusion, profibrogenic mesenchymal cells play an important role in the development of intestinal fibrosis in this chronic DSS-induced colitis model.

DOI： 10.1111/j.1440-1827.2011.02647.x

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記述言語：日本語   出版者・発行元：特定非営利活動法人 日本歯周病学会  

DOI： 10.14833/amjsp.2011s.0.96.0

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その他リンク： http://search.jamas.or.jp/link/ui/2011257348

記述言語：日本語   出版者・発行元：特定非営利活動法人 日本歯周病学会  

DOI： 10.14833/amjsp.2011s.0.14.0

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記述言語：日本語   出版者・発行元：特定非営利活動法人 日本歯周病学会  

DOI： 10.14833/amjsp.2011s.0.118.0

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1902/jop.2010.100194

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SAGE PUBLICATIONS LTD  

Osteogenic potential of biomaterials used in bone regenerative therapy has been mainly examined in an animal-implantation study. We have here evaluated the applicability of bone scintigraphy in imaging ectopic bone formation, especially its initial phase, by beta-tricalcium phosphate (beta-TCP) particles that were implanted in rat dorsal subcutaneous tissues. In implanted osteogenic osteosarcoma cells used as a positive control, osteoid formation was found by histological examination and bone scintigraphy using (99m)Tc- hydroxymethyl diphosphonate (HMDP) at 2 and 3 weeks post-implantation, respectively, while the microfocus-computed tomography (mu CT) system required further mineralization, which occurred at 4 weeks. Implantation of beta-TCP particles alone induced only faint biomineralization inside the particles, which could be microscopically detected by calcein chelation at 2 weeks post-implantation, but not by other histological examinations (e. g., HE staining) or mu CT. However, the bone scintigraphy successfully detected this microscopic change at 1 week. Implanted hydroxyapatite (HAp) particles alone used as a negative control did not induce mineralization at microscopic levels, and therefore nothing was detected by either calcein chelation or bone scintigraphy. In conclusion, the bone scintigraphic methodology, although exhibiting less quantitation and resolution, would be applicable as a non-invasive, highly sensitive methodology in detecting the initial, microscopic changes associated with mineralization.

DOI： 10.1177/0885328209341845

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: A superporous (85%) hydroxyapatite (HA) block was recently developed to improve osteoconductivity, but it was often not clinically successful when used to treat periodontal osseous defects. The primary purpose of this study is to develop a clinically applicable tissue-engineered bone substitute using this HA block and human alveolar periosteum-derived cells.
Methods: Commercially available superporous HA blocks were acid treated and subjected to a three-dimensional (3D) culture for periosteal cell cultivation. Cells in the pore regions of the treated HA block were observed on the fracture surface by scanning electron microscopy. After osteogenic induction, the cell HA complexes were implanted subcutaneously in nude mice. Osteoid formation was histologically evaluated.
Results: Acid treatment enlarged the interconnections among pores, resulting in the deep penetration of periosteal cells. Under these conditions, cells were maintained for &gt;2 weeks without appreciable cell death in the deep pore regions of the HA block. The cell HA complexes that received in vitro osteogenic induction formed osteoids in pore regions of the treated HA blocks in vivo. In contrast, most pore regions in the non-pretreated, cell-free HA blocks that were evaluated in vivo remained cell free.
Conclusions: Our findings suggest that an acid-treated HA block could function as a better scaffold for the 3D high-density culture of human periosteal cells in vitro, and this cell HA complex had significant osteogenic potential at the site of implantation in vivo. Compared with the cell-free HA block, our cell HA complex using periosteal cells, which are the most accessible for clinical periodontists, showed promising results as a bone substitute in periodontal regenerative therapy. J Periodontal 2010;81:420-427.

DOI： 10.1902/jop.2009.090523

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

Our animal implantation studies have demonstrated that, after osteogenic processing, cultured human periosteal sheets form osteoid tissue ectopically without the aid of conventional scaffolding materials. To improve the osteogenic activity of these periosteal sheets, we have tested the effects of including a scaffold made of salmon collagen-coated ePTFE mesh. Periosteal sheets were produced with minimal manipulation without enzymatic digestion. Outgrown cells penetrated into the coated mesh fiber networks to form complex multicellular layers and increased expression of alkaline phosphatase activity in response to the osteoinduction. In vitro mineralization was notably enhanced in the original tissue segment regions, but numerous micro-mineral deposits were also formed on the coated-fiber networks. When implanted subcutaneously into nude mice, periosteal sheets efficiently form osteoid around the mineral deposits. These findings suggest that the intricate three-dimensional mesh composed of collagen-coated fibers substantially augmented the osteogenic activity of human periosteal sheets both in vitro and in vivo.

DOI： 10.1007/s10856-009-3896-9

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：JAPANESE SOCIETY OF PERIODONTOLOGY  

DOI： 10.2329/perio.52.3

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その他リンク： http://search.jamas.or.jp/link/ui/2010198825

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Our recent clinical studies have demonstrated that autologous implantation of human cultured periosteal (hCP) sheets in combination with porous hydroxylapatite (HAp) particles at the site of periodontal bone defects strikingly facilitates tissue regeneration. To better understand how the hCP sheet functions at the implantation site, we have now examined its biochemical and morphological characteristics in vitro and its ectopic osteoinductivity in nude mice. Cultured human periosteal tissue segments produced periosteal cells that migrated out from the central region within 4-8 days and grew more rapidly with longer culture times. Alkaline phosphatase activity increased in parallel with actual osteoblastic induction. Cytokine array assays demonstrated that osteoblastic induction downregulated IL-6 and thrombopoietin, but upregulated IL-8, IL-13, IGF-I and IGFBP-2 in hCP sheets. When differentiated hCP sheets were implanted alone, areas of osteoid and mineralized tissue were formed within 2 weeks, but non-induced, immature hCP sheets did not produce much mineralization. These findings suggest that mature hCP sheets potentially function not only as seeds of ectopic bone formation without the need of synthetic tissue scaffolds, but also as living drugdelivery systems, to influence cells near implantation sites by producing several important cytokines. These two major characteristics indicate that a mature hCP sheet is a promising osteoinductive biomaterial, even without conventional scaffolds for periodontal regenerative therapy. Copyright © 2009 John Wiley &amp
Sons, Ltd.

DOI： 10.1002/term.156

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.11242/dentalradiology.49.33

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：社団法人 日本口腔外科学会  

Based on recent advances in tissue engineering, we have developed three individual therapeuticmethodologies for regeneration of periodontal defects. (1) Human cultured gingival epithelial cell sheets couldbe applied as a promising graft "material" to treatment of chronic desquamative gingivitis. (2) Cultured gingivaldermal substitute could contribute to accelerated recovery of vertical and horizontal gingival recession. (3)Cultured periosteal cell sheets, which are demonstrated to express osteogenic activity in vitro and in vivo animalstudies, could regenerate alveolar bone defects in combination with platelet-rich plasma and porous hydroxapatiteparticles. Finally, current and next-generation technology in tissue engineering and periodontal regenerative medicineshould be discussed.

DOI： 10.5794/jjoms.55.432

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その他リンク： https://jlc.jst.go.jp/DN/JALC/00338247696?from=CiNii

記述言語：日本語   出版者・発行元：特定非営利活動法人 日本歯周病学会  

DOI： 10.14833/amjsp.2009f.0.88.0

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: The aim of the present controlled clinical study was to compare the clinical response of human cultured periosteum (HCP) sheets in combination with platelet-rich plasma (PRP) and porous hydroxyapatite (HA) granules to a mixture of PRP and HA in the treatment of human infrabony periodontal defects.
Methods: Thirty interproximal infrabony osseous defects in 30 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. The subjects were randomly assigned to the test group (HCP sheets combined with PRP and HA) or the control group (PRP with HA). Clinical and radiographic measurements were made at baseline and the 12-month post-surgical evaluation.
Results: Compared to baseline, the 12-month results indicated that both treatment modalities resulted in statistically significant changes (P&lt;0.01) in the gingival index, bleeding on probing, probing depth, clinical attachment level, and radiographic infrabony defect depth. Compared to the control group, the test group exhibited a statistically significantly more favorable change in clinical attachment gain (3.9 +/- 1.6 mm versus 2.7 +/- 1.3 mm; P&lt;0.05), vertical relative attachment gain (83.5% +/- 31.7% versus 55.0% +/- 21.9%; P &lt;0.05), and radiographic infrabony defect fill (4.9 +/- 1.2 mm versus 3.2 +/- 1.1 mm; P&lt;0.01).
Conclusions: Compared to PRP with HA, treatment with a combination of HCP sheets, PRP, and HA led to a significantly more favorable clinical improvement in infrabony periodontal defects. A factor likely contributing to these favorable clinical results is the presence of osteogenic cells in the HCP sheets, which provided greater regeneration potential.

DOI： 10.1902/jop.2008.070518

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: Periodontitis, similar to many other known inflammatory diseases, is thought to increase adenosine triphosphate (ATP) levels in extracellular spaces. Extracellular ATP acts on specific receptors to modulate the proliferation of various cell types. Platelet-rich plasma (PRP) contains high levels of ectonucleotidase activity capable of degrading ATP. The aim of this study was to investigate the effects of ATP on the proliferation of human periodontal ligament (PDL) cells and how these effects are altered by ectonucleotidases.
Methods: PDL cells were derived from healthy young volunteers. ATP content and DNA systhesis were quantified by a bioluminescence and an enzyme-linked immunosorbent assay, respectively. CD39 and p21(WAF1/cip1) expression was analyzed by Western blot. Apoptosis was evaluated by caspase-3/7 activity, terminal deoxynucleotidyl transferase (TST)-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) activity, annexin-V-binding, and DNA fragmentation.
Results: CD38 and ectonucleotidase-like activity were found in PDL cells and serum, respectively. Because less CD39 is expressed in freshly plated cells, both exongenous ATP and ATP gamma S, a slowly hydrolysable analog, inhibited cell proliferation under low serum condition. ATP upregulated p21(WAF1/cip1), an inhibitor of cell-cycle progression, whereas ATp gamma S induced capase-dependent apoptosis. Either upregulated of CD39 or added serum rescued cells from the cytostatic actions of exogenous ATP.
Conclusions: In PDL cells expressing low CD39 levels, both ATP and ATP gamma S inhibited proliferation but by different mechanisms. ATP-induced growth arrest suggests that periodontal tissue regeneration is often supressed at the site of injury. Futhermore, added ectonucleotidases protected PDL cells from ATP's cytostatic actions, suggesting that ectonucleotidase-rich PRP augments the regenerative actions of its constituent growth factors by protecting against exogenous ATP at clinical sites.

DOI： 10.1902/jop.2007.060283

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: In addition to its prominent role in liver regeneration, hepatocyte growth factor (HGF) is now generally thought to be produced by mesenchymal cells to promote the regeneration of epithelial tissue by a paracrine mechanism. However, it is not known how or if HGF could be involved in the regeneration of periodontal tissues. The purpose of this study was to characterize the ability of normal human periodontal ligament (PDL) cells to produce or respond to HGF.
Methods: PDL cells derived from healthy young volunteers were used from passages four through 10. HGF receptors were detected both by immunocytochemical staining and Western-blotting analysis. Both DNA synthesis (by bromo-deoxyuridine [BrdU]-incorporation) and secreted HGF were quantified by enzyme-linked immunosorbent assays. Mitogen -activated protein kinase (MAPK) phosphorylation was also analyzed by Western blot.
Results: Despite the immunocytochemical demonstration of HGF receptor protein in the cytoplasm and on the plasma membrane of PDL cells, exogenous recombinant human HGF did not exert the mitogenic effects expected. As reported for other mesenchymal cells, PDL cells were found to secrete HGF. Treatments with neutralizing anti-HGF antibody significantly suppressed constitutive PDL cell proliferation and sustained the receptor protein at higher levels than in non-treated cells. Under these conditions, exogenous HGF rapidly phosphorylated extracellular signal-regulated kinase (ERK), an action linked to the cell proliferation and downregulation of cell-surface receptors.
Conclusions: Unlike other known mesenchymal or epithelial cells, these findings suggest that normal PDL cells from young donors possess a constitutive HGF/receptor autocrine loop that normally regulates their replacement self-proliferation but reduces sensitivity to exogenously applied HGF by acute receptor downregulation.

DOI： 10.1902/jop.2006.060031

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: The aim of the present controlled clinical study was to compare platelet-rich plasma (PRP) combined with a biodegradable ceramic, porous hydroxyapatite (HA) with a mixture of HA and saline in the treatment of human intrabony defects.
Methods: Seventy interproximal intrabony osseous defects in 70 healthy, non-smoking subjects diagnosed with chronic periodontitis were included in this study. Thirty-five subjects each were randomly assigned to either the test group (PRP and HA) or control group (HA with saline). Clinical and radiographic measurements were determined at baseline and the 12-month evaluation.
Results: When compared to baseline, the 12-month results indicated that, while both treatment modalities resulted in significant changes in all clinical parameters (gingival index, bleeding on probing, probing depth, clinical attachment level, and intrabony defect fill; P &lt; 0.001), the test group exhibited statistically significant changes compared to the control sites in probing depth reduction: 4.7 +/- 1.6 mm versus 3.7 +/- 2.0 mm (P &lt; 0.05); clinical attachment gain: 3.4 +/- 1.7 mm versus 2.0 +/- 1.2 mm (P &lt; 0.001); and vertical relative attachment gain: 70.3% +/- 23.4% versus 45.5% +/- 29.4% (P &lt; 0.001).
Conclusion: Treatment with a combination of PRP and HA compared to HA with saline led to a significantly more favorable clinical improvement in intrabony periodontal defects.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC IN VITRO BIOLOGY  

Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor beta 1 (TGF-beta 1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous Studies, we demonstrated that PRP mimics TGF-beta 1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type 1 collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult Volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells IRA failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: Platelet-rich plasma (PRP) is a fraction of plasma, in which platelet-derived growth factor (PDGF) and transforming growth factor-P (TGF-beta) are thought to be concentrated. It is plausible that topically-applied PRP up-regulates cellular activity and subsequently promotes periodontal regeneration in vivo. However, the concentrations of these growth factors in PRP have not been specifically determined and the biological effects of PRP at the cellular and molecular levels have not been determined.
Methods: PRP obtained from 20 healthy subjects was prepared from plasma by centrifugation. These PRP preparations were immediately subjected to an evaluation for PDGF-AB and TGF-beta1 using enzyme-linked immunosorbent assay (ELISA) kits. The biological effects of the PRP preparations were evaluated on osteoblastic, epithelial, fibroblastic, and periodontal ligament cells. Cellular mitogenic activity was evaluated by counting cell numbers or evaluating 5 - bromodeoxyuridine (BrdU) incorporation. Expression of alkaline phosphatase (ALP) was immunocytochemically evaluated.
Results: In the PRP preparations, platelets were concentrated up to 70.9 x 10(4) cells/mul (283.4% of the unconcentrated plasma). The levels of PDGF-AB and TGF-beta1 were also concentrated up to 182.0 ng/ml (440.6%) and 140.9 ng/ml (346.6%), respectively. Scatter plots revealed significant correlations between platelet counts and levels of these growth factors. PRP stimulated osteoblastic DNA synthesis and cell division (138% of control), with simultaneous down-regulation of ALP, but suppressed epithelial cell division (80% of control). PRP also stimulated DNA synthesis in gingival fibroblasts and periodontal ligament cells.
Conclusions: These data demonstrated that both PDGF-AB and TGF-beta1 were highly concentrated in the PRP preparations. It is suggested PRP modulates cell proliferation in a cell type-specific manner similar to what has been observed with TGF-beta1. Since synchronized behavior of related cell types is thought to be required for successful periodontal regeneration, it is further suggested these cell type-specific actions may be beneficial for periodontal regenerative therapy.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC IN VITRO BIOLOGY  

The stimulatory effects of sodium fluoride (NaF) on bone formation have been explained solely by its activation of osteoblasts. However, whether and how NaF acts on the osteoclast lineage is poorly understood. We previously found that NaF differentiates HL-60 cells to granulocytic cells. To further test this action, we have employed here primary cultures of progenitor cells derived from murine bone marrow. NaF at subtoxic concentrations (&lt;0.5 mM) significantly up-regulated activities of several intracellular enzymes (lactate dehydrogenase, beta-glucuronidase, acid phosphatase), cellular reduction of nitroblue tetrazolium, and nitric oxide (NO) production; which are all accepted as general differentiation markers. NaF (&lt;0.5 mM) also up-regulated granulocyte-specific markers (chloroacetate esterase, cell surface antigens [Mac-1, Gr-1]) but not any of the monocyte-specific markers (nonspecific esterase, cell surface antigens [F4/80, MOMA-2]). Although other general differentiation markers (phagocytosis, adhesion, appearance, nuclear:cytoplasmic ratio) were not appreciably influenced by NaF. essentially in support of our previous data from HL-60 cells, the present findings suggest that NaF induces early differentiation of bone marrow hemopoietic progenitor cells along the granulocytic pathway but not the monocytic pathway that is linked to osteoclast formation. Therefore, in addition to its potent stimulatory effects on osteoblastic bone formation, NaF applied to patients with osteoporosis could be expected to indirectly reduce osteoclastic bone resorption.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL MUNKSGAARD  

We have previously demonstrated that porcine enamel matrix derivative (EMD) contains TGF-beta1 (or a TGF-beta-like substance). and that EMD rapidly translocates smad2, which is an effector of the TGF-beta signaling pathway. into the nucleus and modulates the proliferation of both human gingival fibroblastic and oral epitheiial cells in a cell type-specific manner. To investigate the involvement of TGF-beta in the growth modulatory action of EMD, two approaches have been used in the present study: i) a neutralizing anti-TGF-beta antibody to block EMD action, and ii) authentic porcine TGF-beta1 to compare with EMD. Both in epithelial and fibroblastic cells, TGF-beta1 closely mimicked EMD in nuclear accumulation of smad2, phosphorylation of MAP kinase family members, and consequent cell type-specific growth modulation, Anti-TGF-beta antibody, at levels which completely blocked TGF-beta1-induced smad2 translocation. strongly blocked EMD-induced smad2 translocation. This antibody also blocked other actions of EMD in epithelial cells, i.e. p38-MAP kinase (p38-K) phosphorylation, p21(WAF1/cip1) expression. and inhibition of DNA synthesis. In support of our previous proposal, these data suggest that TGF-beta1 (or a TGF-beta-like substance), which is delivered as a principal bioactive factor in EMD, inhibits epithelial cell proliferation probably by a smad2- mediated, p21(WAF1/cip1)-dependent mechanism. However, the same neutralizing antibody failed to convincingly block EMD-induced fibroblastic proliferation, which suggests that EMD may contain additional unidentified mitogenic factor(s), which act in combination with TGF-beta to fully stimulate fibroblastic proliferation.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MUNKSGAARD INT PUBL LTD  

During surgical treatment of periodontal disease, enamel matrix derivative (EMD) is topically applied as a substitute for extracellular matrix in order to facilitate regeneration of damaged periodontal tissue. However, the mechanism for EMD action is poorly understood, We have now examined the effects of EMD on the proliferation of oral epithelial (SCC25) cells in vitro. After 3 days of treatments, EMD (25-100 mu g/ml) dose-dependently inhibited cell division and concomitantly arrested cell cycle at the G1 phase. Prior to this inhibition, EMD significantly up-regulated p21(WAF1/cip1), a cyclin-dependent kinase inhibitor, induced G1-arrest, and inhibited DNA synthesis. In addition, EMD down-regulated expression of cytokeratin-18 (CK18) protein, which was most due to decreased production, but less to increased degradation. However, EMD did not discernibly increase the number of apoptotic cells over 8 days of treatment. These findings indicate (I)that EMD acts as a cytostatic agent, rather than a cytotoxic agent, on epithelial cells, and (2) that this anti-proliferative action is probably due to p21(WAF1/cip1)- mediated G1-arrest. Furthermore, our in vitro cellular data clearly verify and provide an explanation for the clinical observation that EMD application suppresses the down-growth of junctional epithelium onto dental root surfaces, a process that frequently interferes with the formation of new connective tissue attachments.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MUNKSGAARD INT PUBL LTD  

In many peripheral tissues. calcitonin gene-related peptide (CGRP) is released from peptidergic sensory nerve fibres and acts like a growth factor during tissue development and regeneration. However, the ability of CGRP to influence gingival tissue has not been studied. To address this question, we have now examined the effects of CGRP on the proliferation of human gingival fibroblasts (Gin-l) in vitro. Gin-1 cells have approximately 3100 specific CGRP-binding sites with a Kd of 38.6 pM on their surface. Treatment with CGRP (0.1-100 nM) significantly stimulated cell proliferation in a dose-dependent manner, with maximal effects at 1-10 nM CGRP after 2 d. As one early cellular response to CORP, p44-MAPK protein (also known as the extracellular signal response kinase [ERK]) was tyrosine- and threonine-phosphorylated within 2 min, and this phosphorylation was sustained for at least 1 h. The dose-response curve of MAPK activation was very similar to that observed for CGRP's stimulation of cell proliferation. In addition, CGRP's activation of MAPK stimulated its ability to phosphorylate the Elk-l transcription factor. When cells were pretreated with PD98059, a selective inhibitor of MAPK kinase (also known as MEK), CGRP not only failed to induce phosphorylation of MAPK but also failed to stimulate Gin-1 cell proliferation. Our present data indicate that CGRP rapidly activates the MAPK signalling pathway, an effect which consequently stimulates the proliferation of gingival fibroblasts. Our data demonstrate specific cellular responses to CGRP by gingival fibroblasts and support the possibility that CGRP acts as a targeted local factor in the regulation of development, generation and/or regeneration of gingival tissues.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca-24- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (less than or equal to 10(-6)M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDP beta s). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca2+- and G protein-mediated pathways un UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like: cells that contrasts with the simpler regulatory mechanisms found in A431 cells. (C) 1999 Elsevier Science Ltd. All rights reserved.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ENDOCRINE SOC  

In previous studies, we have shown that calcitonin gene-related peptide (CGRP) acutely inhibits Ca-45(2+) uptake in osteoblastic UMR106 cells, and we have proposed that ATP-sensitive potassium (K-ATP) channels are involved in mediating this action of CGRP. To directly test this proposed mechanism, we have now examined the effects of CGRP on both membrane potential (Em) and K+ mobilization in UMR106 cells, using specific fluorometric dye assays. CGRP (0.01-100 nM) induced membrane hyperpolarization in a dose-dependent manner, with a half maximal effect (ED50) at approximately 0.2 nM and a maximal effect at 100 nM. Both pinacidil (Pina; a K-ATP channel opener) and forskolin (FSK) induced similar membrane hyperpolarization, but the actions of these three agents could be easily distinguished: both CGRP and Pina actions were well antagonized by glibenclamide (Glib; a selective K-ATP channel blocker), whereas FSK action was strongly attenuated only by tetraethylammonium (a K-Ca channel blocker) or compound H-89 (an inhibitor of cAMP-dependent protein kinases). Cells pretreated with Pina no longer responded to CGRP, but they could still respond to FSK; furthermore, pretreatment with FSK failed to block successive treatment with either CGRP or Pina. In parallel with observed changes in Em, CGRP (0.01-100 nM) decreased intracellular K+ concentrations ([K+](i)) in a dose-dependent manner, with an ED50 identical to that obtained for alterations in Em. This action of CGRP was sensitive to Glib and had only slight sensitivity to tetraethylammonium; this CGRP effect was mimicked by Pina but not by FSK. Interestingly, CGRP significantly elicited changes in cell shape by a Glib-sensitive mechanism that included notable decreases in cross-sectional cytoplasmic area. These observations strongly support our proposal that CGRP primarily stimulates K+ efflux via activation of K-ATP channels and thereby induces membrane hyperpolarization in UMR106 cells. Furthermore, our data also suggest that this cascade of initial cellular events may result in rapid changes in cell morphology and decreases in cellular area of the type that are thought to act as triggers for proliferation and/or differentiation in many cellular phenotypes.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL SCIENCE INC  

Sodium fluoride (NaF) is known to stimulate osteoblastic bone formation, but little attention has been given to the possibility that NaF also affects bone resorption and the differentiation of osteoclastic progenitor cells, Wen human promyelocytic HL-60 cells were treated with NaF (0.5 mM, 0-4 days), cell proliferation was inhibited, and the addition of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) (10 nM, 0-4 days) augmented this antiproliferative effect. NaF increased cel)dar reduction of nitroblue tetrazolium (NET), and this effect was strongly augmented by 1,25(OH)(2)D-3. In addition, NaF produced marked changes in cellular morphology, increased cellular adhesion to plastic, reduced the nuclear/cytoplasmic ratio, and increased cellular expression of chloroacetate esterase, but failed to alter cellular nonspecific esterase activity. Furthermore, NaF increased expression of CD11b and CD66b, and this stimulation was enhanced by adding 1,25(OH)(2)D-3. The sum of these changes in classical promyelocytic cellular indices suggest: (1) that NaF stimulates the early stages of HL-60 differentiation toward a granulocyte-like cell and (2) that 1,25(OH)(2)D-3 promotes these actions of NaF. Additional experiments aimed at further understanding the NaF-induced conversion of HL-60 cells identified further changes. NaF also increased cellular production of prostaglandin E(2) (PGE(2)) and nitric oxide (NO) and induced expression of inducible nitric oxide synthase (iNOS); 1,25(OH)(2)D-3 once again augmented these NaF-induced effects. Similarly, NaF stimulated the production of interleukin 1 alpha (IL-1 alpha). IL-6, and tumor necrosis factor-alpha and 1,25(OH)(2)D-3 again strongly enhanced these effects. Indomethacin completely blocked stimulation of NET reduction, NO production, and iNOS expression induced by NaF plus 1,25(OH)(2)D-3; adding exogenous PGE(2) (0.1-10 ng/ml) to these indomethacin-blocked cultures dose-dependently restored NO production. These additional findings together with the observed slow onset (24-18 h) of NaF and 1,25(OH)(2)D-3 interaction strongly suggest that 1,25(OH)(2)D-3 acts as a cofactor with NaF primarily through interaction with an endogenous NaF-induced cyclo-oxygenase product(s), quite possibly PGE(2) itself. Such a mechanism for NaF and 125(OPI)(2)D-3 interaction would be strongly analogous to the interaction we have recently demonstrated between 1,25(OH)(2)D-3 and PGE(1) on the differentiation of HL-60 cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER VERLAG  

Human promyelocytic HL-60 cells can be induced by biochemical agents to differentiate in vitro towards divergent types of myelomonocytic cells. It has been reported that prostaglandin E(1) (PGE(1)) can induce granulocytic differentiation and that 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) can induce monocytic differentiation. We have now examined the effects of these compounds, both alone and in combination, on HL-60 cell differentiation. PGE(1) (1 mu g/ml) or 1,25(OH)(2)D-3 (10 nM) each inhibited cell proliferation over 48-96 hours of treatment, but combined treatment with both agents was necessary to produce a strong inhibition. The percentage of HL-60 cells that can reduce nitroblue tetrazolium (NBT) (a characteristic index of early monocytic or granulocytic differentiation) increased 13-fold within 72 hours of PGE(1) treatment, and 1,25(OH)(2)D-3 produced a fivefold stimulation. However, combined treatment (PGE(1) plus 1,25(OH)(2)D-3) produced a dramatic 35-fold increase. HL-60 cells did not produce significant levels of nitric oxide (NO) before 48 hours in culture, and treatment with PGE(1) or 1,25(OH)(2)D-3 did not significantly increase cellular NO elaboration over control levels. However, combined treatment produced a striking 12-fold increase over control levels. Similarly, combined treatment was necessary to obtain the maximal time-dependent stimulation of cellular lactate dehydrogenase (LDH) activity (a marker of granulocytic differentiation) as well as acid phosphatase (ACP) activity. During this same period of time, PGE(1), but not 1,25(OH)(2)D-3, markedly stimulated cellular elaboration of interleukin (IL)-1 alpha, IL-6, and tumor necrosis factor (TNF)-alpha, and 1,25(OH)(2)D-3 cotreatment strongly augmented these effects. Thus, combined treatment with 1,25(OH)(2)D-3 plus PGE(1) generally augmented the apparent conversion of these cells, producing synergistic (multiplicative) or additive effects. Furthermore, PGE(1) induced within 48 hours the more general phenotypic changes classically associated with the differentiation of these cells: increased expression of chloroacetate esterase (ChAE) (a granulocytic marker), decreases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in morphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)(2)D-3 had little effect itself on most of these parameters, but augmented the morphological changes induced by PGE, treatment. Within 48 hours, the ability of these cells to reduce the tetrazolium salt WST-1, a general measure of cellular metabolic activity, was increased by PGE(1), but not by 1,25(OH)(2)D-3; however, the combination of 1,25(OH)(2)D-3 and PGE(1) again produced the strongest stimulation. Similarly, only PGE(1) significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. In summary, our findings suggest that PGE(1), not 1,25(OH)(2)D-3, is sufficient to promote rapid in vitro differentiation of HL-60 cells along the granulocytic pathway; however, the PGE(1)-induced conversion of these cells is markedly augmented by cotreatment with 1,25(OH)(2)D-3.
In addition, these converted HL-60 cells preferentially utilize the glycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mature granulocytes.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) produce a dose-dependent stimulation in the rate of cell division in a rat clonal dental pulp-cell line (RDP 4-1). To elucidate the initial mitogen-induced cellular events that may mediate mitogenic action, the effects of EGF and IGF-I on cellular protein tyrosine phosphorylation were examined. In a dose-dependent manner, EGF (1-100 ng/ml) transiently stimulated tyrosine phosphorylation in four major proteins with apparent molecular weights of 220, 180, 140 and 120 kDa, and in five other more minor proteins (90, 80, 65, 55 and 44 kDa). IGF-I (1-100 ng/ml) dose-dependently stimulated the tyrosine phosphorylation of 160- and 140-kDa proteins, and had a smaller effect on the 80-, 65- and 44 kDa proteins. In contrast to the action of EGF, IGF-I-induced tyrosine phosphorylation was sustained for more than 60 min, particularly that of the 160-kDa phosphoprotein. From the results of specific immunoprecipitation/Western-blot analyses, the 180-kDa EGF-sensitive protein could be identified as the EGF receptor (EGF-R). Among the IGF-I-sensitive pulp cell proteins, the 160-kDa protein was identified as insulin-receptor substrate-1. Both mitogenic treatments stimulated the tyrosine phosphorylation of a weak, 44-kDa protein, which we have identified as the extracellular signal-regulated kinase-1. Despite the presence of phosphoproteins of the correct size, neither the IGF-I receptor (IGF-I-R) nor the phospholipase C gamma-isoform could be identified as tyrosine kinase substrates in either treatment. Pretreatment with the tyrosine kinase inhibitor genistein (20 mu g/ml) significantly inhibited EGF- and IGF-I-induced tyrosine phosphorylation in permeabilized RDP 4-1 cells, and the tyrosine phosphatase inhibitor orthovanadate (1 mM) significantly prolonged the duration of the mitogen-stimulated tyrosine phosphorylation in both intact or permeabilized cells. Phorbol 12-myristate 13-acetate (100 nM), which activates protein kinase C (PKC), inhibited the tyrosine phosphorylation induced by either growth factor. This action was blocked by pretreatment with staurosporine (200 nM, 15 min), a selective PKC inhibitor. However, neither removing external Ca2+ with EGTA (1 mM) nor inducing Ca2+ influx with A23187 ionophore (2 mu M) significantly altered EGF- or IGF-I-induced phosphorylation. These findings strongly suggest that authentic EGF-R and IGF-I-R on RDP 4-1 cells are coupled to complex, tyrosine kinase-mediated, intracellular signalling systems that are sensitive to a PKC-dependent mechanism. EGF- and IGF-I-induced tyrosine phosphorylation cascades may have important roles in vivo in the regulation of dental pulp-cell proliferation and ultimately may affect dentine formation.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE PUBL CO INC  

Calcitonin gene-related peptide (CGRP) was examined for its effects on intracellular free Ca2+ concentration ([Ca2+](i)) in UMR 106 osteoblast-like cells, Cells loaded with the Ca2+ dye FURA-2 dose-dependently responded to CGRP (1-100 nM) with transient two-fold increases in [Ca2+](i), An intracellular source for this Ca2+ transient was suggested by the failure of membrane depolarization with high extracellular K+ or acute depletion of extracellular Ca2+ ([Ca2+](e) with EGTA to attenuate this response, After cells were incubated for 45 min with 0.1 mM extracellular Ca2+ to deplete intracellular Ca2+ stores, CGRP produced a 25-30% decrease in [Ca2+](i) rather than a transient increase, This calcium decrease was mimicked by membrane depolarization or by pinacidil, a specific activator of adenosine triphosphate (ATP)-sensitive potassium (K-ATP channels, and blocked by glybenclamide, a specific blocker of K-ATP channels. Our data suggest that CGRP has diverse Ca2+ regulatory effects in UMR 106 cells, mobilizing Ca2+ from intracellular stores via classical signaling while possibly promoting cellular Ca2+ efflux or inhibiting uptake through voltage-dependent Ca2+ channels via K-ATP-mediated hyperpolarization.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER VERLAG  

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) was recently shown to promote maturation of 5-fluorouracil (5FU)-treated bone marrow cells by up-regulating macrophage-colony stimulating factor (M-CSF) receptors in the presence of interleukin 1alpha (IL-1alpha). In order to reveal how 1,25(OH)2D3 interacts with colony-stimulating factors and regulates the differentiation of bone marrow progenitor cell populations, in the present study, natural bone marrow cells were isolated from untreated mice and used in alpha-minimum essential medium supplemented with 20% heat-inactivated horse serum without added appropriate cytokines. Under the conditions, cells spontaneously differentiated gradually with days of culture, as assessed by expression of macrophage differentiation antigens such as Mac-1 (CD11b) and F4/80. Both M-CSF and granulocyte macrophage-colony stimulating factor (GM-CSF) induced only Mac-1 antigen expression. Simultaneous treatment with M-CSF and 1,25(OH)2D3 enhanced the M-CSF's effect on expression of both antigens, although 1,25(OH)2D3 per se has no effect on the expression for up to 11 days. In addition, successive treatment with 1,25(OH)2D3 and M-CSF or GM-CSF dramatically enhanced expression of both antigens or Mac-1 antigen, respectively. Similarly, both simultaneous and successive treatment with 1,25(OH)2D3 and M-CSF significantly enhanced phagocytic activity and H2O2 production, whereas successive treatment with 1,25(OH)2D3 and GMCSF significantly enhanced only phagocytic activity. Enzyme-histochemical study demonstrated that cells treated simultaneously or successively with 1,25(OH)2D3 and M-CSF were strongly positive for nonspecific esterase (NSE), a macrophage-specific marker, and that simultaneous or successive treatment with 1,25(OH)2D3 and GM-CSF yielded cells strongly positive for NSE or for chloroacetate esterase (ChAE), a granulocyte-specific marker, respectively. These findings suggest that 1,25(OH)2D3 primes bone marrow progenitor cell populations not only to M-CSF but also to GM-CSF and thereby accelerates the CSFs-dependent differentiation of the cells to the macrophage or granulocyte.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

The cloned pulp-cell line RDP4-1 increases cAMP production, hydrolyses phosphoinositide (PI) and mobilizes calcium in response to prostaglandin E2. (PGE2) and PGF2-alpha. The effect of bradykinin (BK) on intracellular signalling systems and DNA synthesis was studied in these cells. BK (10 muM) transiently increased cytoplasmic free calcium ([Ca2+]i) both in the presence and absence of external calcium. After stimulation with BK (10 muM), cells did not respond significantly to PGE2 (0.5 mug/ml). Pretreatment with indomethacin (30 muM) inhibited the [Ca2+]i increment by BK (10 muM), but not by the subsequent addition of PGE2 (0.5 mug/ml). Also, pretreatment with PGE2 (0.5 mug/ml) blocked the action of BK (10 muM). BK (0.1-100 muM) stimulated PI hydrolysis and cAMP production in a dose-dependent manner. Both the PI and the cAMP responses were inhibited by indomethacin (30 muM), as was the calcium response. BK (0.01-10 muM) also stimulated release of arachidonic acid and its metabolites dose-dependently. However, prolonged exposure to BK in serum-deficient medium did not exert any effect on DNA synthesis. RDP 4-1 cells, therefore, appear to respond to BK with increased cAMP production, PI hydrolysis and calcium mobilization. The inhibition of these effects of BK by indomethacin raises the possibility that cyclo-oxygenase product(s), especially PGE2 or PGE2-like compounds, may be responsible for evoking these effects. These results indicate that BK may stimulate or modulate cell metabolism in the dental pulp.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

The purpose of this study is to investigate the involvement of protein kinase C (PKC) in prostaglandin E2 (PGE2)-stimulated cAMP production of two macrophage-like cell lines (G3 and XC). XC cells are thought to be placed at a more differentiated stage than G3 cells [Orikasa et al. (1991) Cell Immunol. 132, 350-365]. In RPMI 1640 containing 10% (v/v) heat-inactivated foetal calf serum (FCS), in which the cAMP response of both cells to PGE, increased with duration of culture, XC cells showed greater response than G3 cells at 2 days of culture. In alpha-minimum essential medium (alpha-MEM) containing 20% (v/v) heat-inactivated horse serum (HS), the cAMP response of both cells was apparently greater than that in RPMI 1640 containing 10% (v/v) FCS. These cells increased cAMP production also in response to PGE, and PGF2alpha, and the order of potency in increase was PGE1 &gt; PGE2 much greater than PGF2alpha. Interestingly, a short-term (20 min) treatment with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC or staurosporine, a relatively specific inhibitor of PKC, augmented the PGE2-stimulated cAMP production in these cells cultured in alpha-MEM containing 20% (v/v) HS. However, a long-term (24 h) treatment with these compounds did not alter the cAMP response. In G3 cells, PMA appeared more potent than staurosporine in terms of augmentation, whereas in XC cells, the former appeared less potent than the latter. Although the mechanism of this augmentation seems complex and is still unclear, our findings suggest that PKC modulates the regulation of the PGE2-cAMP signalling system, and that the macrophage differentiation may decrease the involvement of PMA-sensitive PKC, but not that of staurosporine-sensitive PKC, in this cAMP system.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

We examined the effect of phorbol 12-myristate 13-acetate (PMA) on release of arachidonic acid (AA) and its metabolites in osteoblastic cells in an attempt to study mechanism of the regulation of phospholipase A2 (PLA2) activity. In the MOB 3-4-F2 cell line, a subclone of the clonal osteoblastic MOB 3-4 cell line, PMA (0.1-100 nM) changed its appearance and increased AA release in a dose- and time-dependent manner, whereas 4-alpha-phorbol 12, 13-didecanoate (4-alpha-PDD) did not show a significant affect on the release. The addition of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, greater-than-or-equal-to 1.5 mM), a Ca2+ chelator, almost completely inhibited the PMA-induced AA release without affecting the intrinsic AA release. Preincubation with staurosporine (5-20 nM), an inhibitor of protein kinase C (PKC), partially (approximately 60%) blocked the AA release. However, 30-min preincubation with H-7 (50-200-mu-M), an inhibitor of PKC, failed to block the AA release. PMA, thus, appeared to stimulate AA release partially by a staurosporine-sensitive mechanism, probably an activation of PKC, in an external Ca2+-dependent manner. On the other hand, MOB 3-4 cells responded to PMA with an increased AA release but not with a drastic change of its shape. Both staurosporine and BAPTA exerted similar inhibitory effects. Prolonged exposure (48 h) to PMA (0.1-10 nM) enhanced DNA synthesis of MOB 3-4-F2 cells, but not MOB 3-4 cells. Taken together with Ca2+-dependence of PLA2, these findings suggest that the PMA-induced release of AA and its metabolites in both MOB 3-4 and MOB 3-4-F2 cells is due to activation of PLA2 by both a staurosporine-sensitive mechanism, probably an activation of PKC, and a staurosporine-insensitive mechanism. In addition, the comparison between the MOB 3-4 cell line and its subclone, MOB 3-4-F2, may provide a useful system for studying the physiological or pathological role of PKC in bone-derived cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MUNKSGAARD INT PUBL LTD  

In attempt to study the mechanism of F--induced, osteoblast-mediated bone formation, we tried to show the characteristics of Al-F complex-induced mitogenesis in osteoblastic cells. The MOB 3-4-F2 cell line, an osteoblast-like cell line derived from neonatal mouse calvaria, responded to F- (1-2 mM) combined with Al3+ and epidermal growth factor (EGF, 0.01-100 ng/ml) with increased DNA synthesis. Of the several types of Al-F complexes, AlF4- is thought to act as a mitogenic factor. On the other hand, NaF at high concentrations (&gt; 2 mM) markedly decreased cell viability. The AlF4--stimulated DNA synthesis at least with a delay of 48 hr, while EGF stimulated DNA synthesis within a few hours (4-6 hr). Both 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine, inhibitors of protein kinase C (PKC), further enhanced DNA synthesis in AlF4--treated cells, whereas 12-O-tetradecanoyl-13-acetate (TPA), an activator of PKC, decreased the DNA synthesis. In EGF-treated cells, staurosporine and TPA, but not H-7, decreased DNA synthesis. In addition, indomethacin, an inhibitor of cyclooxygenase, partly inhibited the EGF-induced mitogenesis, which, however, was restored by addition of PGE2. AlF4-, as well as EGF, stimulated the release of arachidonic acid and its metabolites. Indomethacin failed to inhibit the AlF4--induced mitogenesis. Thus, the mitogenic response of MOB 3-4-F2 cells to F- in the presence of Al3+ had the following characteristics: (1) it was effective over a narrow range, (2) it had a slow onset, (3) included a PKC-sensitive mechanism and (4) a PG(E2)-independent mechanism. In contrast, a wide range of EGF concentrations rapidly stimulated DNA synthesis by a PKC-sensitive, PG(E2)-dependent mechanism in these cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER VERLAG  

A clonal osteoblast-like cell line, MOB 3-4, increased cAMP production in response to prostaglandin E2 (PGE2) (5-500 ng/ml). The purpose of this study was to show the effects of tumorpromoting phorbol ester (e.g., 12-O-tetradecanoylphorbol 13-acetate, TPA) on basal and PGE2-stimulated cAMP production and the affinity of PGE2 receptors in the cells. Pretreatment with TPA (1 nM-10-mu-M) for 30 minutes increased basal cAMP production, whereas it markedly reduced the PGE2-stimulated cAMP production in the presence of 0.1 mM isobuthylmethyl xanthine. Both the TPA increase and reduction were dose- and time-dependent. However, TPA exerted no effect on forskolinor cholera toxin-stimulated cAMP production. Copretreatment with TPA and H-7, an inhibitor of protein kinase C (PKC), prevented the TPA-induced increase in basal cAMP production, whereas it did not prevent the reduction of the PGE2-stimulated cAMP production. On the other hand, TPA (0.1-10 mu-M) decreased H-3-PGE2 binding in a dose- and time-dependent manner. Scatchard analysis revealed that TPA decreased the apparent affinity of PGE2 receptors without effect on their apparent number. In addition, 1-oleoyl-2-acetylglycerol (12.6 mu-M), a synthetic diacylglycerol analog, did not mimic the TPA action on H-3-PGE2 binding. Thus, TPA at relatively high concentrations appeared to increase basal cAMP production by a PKC-mediated mechanism, and it appeared to directly act on and thereby reduce the PGE2-stimulated cAMP production in the clonal osteoblast-like MOB 3-4 cell line.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-LISS  

Prostaglandin E2 (PGE2, 5 ng/ml to 5-mu-g/ml) induced a dose-dependent increase in cAMP accumulation, inositol phosphates (IPs) accumulation, and cytoplasmic free Ca2+ ([Ca2+]i) in a clonal osteoblast-like cell line, MOB 3-4. In contrast, prostaglandin F2-alpha (PGF2-alpha, 5 ng/ml to 5-mu-g/ml) stimulated increases in IPs accumulation and [Ca2+]i without stimulating an increase in cAMP accumulation. Both PGE2 (&gt; 0.5-mu-g/ml) and PGF2-alpha (greater-than-or-equal-to 0.5-mu-g/ml) increased cytoplasmic pH (pHi) from approximately 7.15 to 7.35 in BCECF-loaded cells. A tumor promotor, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nM) also increased pHi without effect on phosphoinositide hydrolysis. Both PGE2-(5-mu-g/ml) and PMA- (100 nM) induced cytoplasmic alkalinization was inhibited by removal of extracellular Na+, or by pretreatment of the cells with amiloride (0.5 mM), an inhibitor of Na+/H+ exchange, or H-7 (100-mu-M), a nonspecific inhibitor of protein kinase C. Thus, MOB 3-4 cells appeared to possess PGE2 receptors and PGF2-alpha receptors: the former are coupled to adenylate cyclase and phospholipase C, and the latter are predominantly coupled to phospholipase C. Also the cells appeared to possess an amiloride-sensitive Na+/H+ exchange activity, which increases pHi in response to PGE2 and PGF2-alpha, as well as to PMA. Long-term (48 hr) exposure of the cells to PGE2 at a high concentration (5-mu-g/ml), but not to PGF2-alpha and PMA, decreased DNA synthesis in the serum-deficient medium. Thus, cytoplasmic alkalinization appeared insufficient for cell replication. At least in MOB 3-4 cells, the inhibitory effect of PGE2 on DNA synthesis may be due to the cAMP messenger system.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

Prostaglandin E2 (PGE2) stimulated cAMP production in the MOB 3-4-F2 cell line, a subclone of the osteoblast-like MOB 3-4 cell line. After being cultured in alpha-minimum essential medium supplemented with 10% heat-inactivated foetal calf serum (HIFCS), cells responded to PGE2 (greater-than-or-equal-to 50 ng/ml) with a small, but significant, increase in cAMP production. This response did not vary with duration of culture. In 2% HIFCS-containing medium, despite their lower basal cAMP level, cells responded to PGE2 (greater-than-or-equal-to 5 ng/ml) with strikingly increased cAMP production. In addition, prolonged culture in this serum-deficient medium enhanced this response. On the other hand, culture of cells in 2% HIFCS-containing medium decreased the apparent number of PGE2 receptors, which was also enhanced by prolonged culture, without effect on their apparent affinity. Their number in 10% HIFCS-containing medium, more than that in 2% HIFCS-containing medium, was almost constant, independent of the culture period. Starvation of MOB 3-4-F2 cells in serum-deficient medium, therefore, appeared to down-regulate PGE2 receptors but increase the cAMP response to PGE2. Moreover, prolonged starvation of cells appeared to facilitate these phenomena. Our findings suggest that cAMP response to PGE2 does not always reflect the number of available PGE2 receptors in the cells.

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCI IRELAND LTD  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A number of studies have shown membrane phospholipid metabolism to have an important role in biological mineralization. We considered the effects of exogenously applied phosphatidic acid (PA), a minor component of membrane phospholipids, on an osteoblast-like cell line, MOB 3-4. Exogenous PA (10-40 μg/ml) raised the level of cytoplasmic free Ca2+ ([Ca2+]i), independent of the level of extracellular Ca2+, in a dose-dependent fashion, and this Ca2+ response to PA gradually fell on serial application of PA. In a dose-dependent manner, exogenous PA also increased inositol 1,4,5-trisphosphate (IP3) accumulation and cytoplasmic pH, but decreased basal cAMP level. This cytoplasmic alkalinization was inhibited by pretreatment with nonspecific protein kinase C (PKC) inhibitors, such as sphingosine or H-7. A long-term incubation with PA increased alkaline phosphatase (ALP) activity and cell proliferation. Exogenous PA thus appeared to increase IP3 accumulation by activating phospholipase C, raise [Ca2+] by releasing Ca2+ from intracellular stores, induce cytoplasmic alkalinization via a PKC-dependent mechanism, and simultaneously decrease basal cAMP level. We suggest that these initial responses may be responsible for the increase in ALP activity and the proliferation of PA-treated MOB 3-4 cells. © 1990.

DOI： 10.1016/0169-6009(90)90049-L

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Abstract: The effect of fluoride on cytoplasmic pH (pHi) in L929 cells was investigated by using a fluorescent pH indicator, BCECF. Fluoride decreased pHi in a dose‐dependent manner. This cytoplasmic acidification was composed of two phases: 1) a rapid decrease in pHi occurring within seconds, and 2) a slow decrease in pHi occurring 1–2 min. after stimulation with fluoride. The phase one decrease in pHi at external pH (pHe) 7.7 was more rapid than that at pHe 6.8, whereas the phase two decrease at pHe 7.7 was slower than that at pHe 6.8. In addition, both in the protein kinase C‐inhibited and depleted cells, the fluoride‐acidified pHi gradually returned to the resting pHi level in phase two, though the initial cytoplasmic acidification (i.e. phase one) was not affected. These results suggest that the fluoride‐induced cytoplasmic acidification is dependent upon pHe and is sustained by the protein kinase C‐dependent Na+/H+ exchange. 1989 Nordic Pharmacological Society

DOI： 10.1111/j.1600-0773.1989.tb00680.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A decrease in the pH of the medium facilitated fluoride (F-) influx but depressed its efflux, which is consistent with the hypothesis that simple diffusion of hydrogen fluoride (HF) contributes to F- migration across the cell membrane. Long-term exposure to F- (&gt
1 mM) induced F- accumulation and, as a result, inhibited cell proliferation. In contrast, short-term exposure to F- (&gt
1 mM), followed by careful washing, did not inhibit cell proliferation but rather stimulated it. Moreover, this stimulatory effect was enhanced by 1 μM Al3+ and was inhibited by 1 μM H-7, a specific inhibitor of protein kinase C. Thus F- may stimulate cell proliferation by activating protein kinase C through GTP-binding protein. The stimulatory effect of F- on cell proliferation, which is usually hidden by its inhibitory effect, can be observed by preventing the accumulation of F- in the cytoplasm. © 1989.

DOI： 10.1016/0003-9969(89)90133-7

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press  

Phosphatidic acid (PA) evoked a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]1) in osteoblasts isolated from neonatal mouse calvaria. This increase was observed in both low (below 150 μM) and high (1.26 mM) Ca2+-containing medium. In contrast, other phospholipids, such as phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, failed to increase [Ca2+]1 in osteoblasts. In high Ca2+-containing medium, A23187 also increased [Ca2+]1 in the cells, but the mode of the change was different from that in the case of PA. These results suggest that PA may induce Ca2+-mediated cellular responses through Ca2+ release from intracellular stores in osteoblasts. © 1988 BY The Journal of Biochemistry.

DOI： 10.1093/oxfordjournals.jbchem.a122309

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The effect of NaF on cytosolic free Ca2+ concentrations ([Ca2+]i) was examined in a clonal osteoblast-like cell line (MOB 3-4) loaded with Fura 2. MOB 3-4 cells in a sparse culture, which exhibited neither alkaline phosphatase (ALP) activity nor the response to parathyroid hormone (PTH), responded to NaF (0.1-10 mM) to increase [Ca2+]i transiently. In contrast, the cells in a dense culture, which exhibited both ALP activity and the response to PTH, responded to NaF (above 4 mM) to increase [Ca2+]i slowly. [Ca2+]i in osteoblasts in primary culture slowly increased in response to both NaF (above 4 mM) and PTH (3 U/ml). Thus, the sensitivity and the response of MOB 3-4 cells to NaF and PTH varied with the culture density, and high culture density matured the cells like osteoblasts in primary culture. These NaF-induced Ca2+ mobilizations were not dependent upon external Ca2+ and were enhanced by Al3+ (1 μM), whereas the PTH-induced Ca2+ mobilizations were due to Ca2+ influx. These results suggest that the maturation of MOB 3-4 cells, dependent upon the culture density, modulates intracellular signal transduction pathways and thereby alters the NaF-induced Ca2+ mobilization, and that the culture density must be taken into consideration in studying Ca2+ mobilization in such an osteoblast-like cells line as MOB 3-4 cell line. © 1988.

DOI： 10.1016/0024-3205(88)90417-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The sensitivity of fibroblasts (L cells) to low concentrations of sodium fluoride (NaF) was examined using the same cell during a series of stimuli. NaF with increasing concentrations up to 1 mM elevated cytosolic free Ca2+ concentrations ([Ca2+]i) in single cells. [Ca2+]i increased within 15 sec of addition of NaF and lowered to basal [Ca2+]i levels quickly. This elevation was observed both in the presence and absence of external Ca2+ and was enhanced by 1 μM Al3+. These results suggest that low concentrations (below 1 mM) of NaF induce Ca2+ release from intracellular Ca2+ stores in single L cells through guanine nucleotide-binding protein (G protein). © 1988.

DOI： 10.1016/0024-3205(88)90557-7

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記述言語：日本語   出版者・発行元：(一社)日本核医学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：WILEY-BLACKWELL  

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記述言語：日本語   出版者・発行元：新潟医学会  

To regenerate periodontal tissue destroyed by chronic periodontitis, autologous cultured periosteal sheets in combination with platelet-rich plasma (PRP) and hydroxyapatite (HA) granules were used as a clinical application. The periosteum specimens were dissected from each patient, and were incubated for approximately six weeks until the sheets were formed. Prior to placement of the cultured periosteal sheets, osseous defects were thoroughly debrided and filled with HA granules in combination with PRP. Standardized clinical and radiographic measurements were determined during follow-up examinations at one, two and three years. Clinical attachment level (CAL) and radiographic infrabony defect depth (IBD) were determined as primary end-point. As secondary end-point, inflammatory indices including plaque index (PI), gingival index (GI) and bleeding on probing (BOP) were determined. Results were averaged for the clinical and radiographic parameters. Taking into account the paired nature of the changes from baseline to one year, and three years in each group, the Wilcoxon signed-rank test was performed. The Mann-Whitney U test was applied to compare clinical and radiographic outcomes between test and control groups at baseline and 1 year post surgery. The null hypothesis was rejected when the risk percentage was < 5% (p<0.05). One year results of this treatment have shown favorable clinical results, and these effects were stable and maintained after three years, when compared to baseline. The cultured periosteal sheets function as a living drug delivery system to favorably influence cellular functions and serves as seeds for ectopic bone formation near the implantation site through production of important growth factors related to periodontal regeneration.

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その他リンク： http://hdl.handle.net/10191/36051

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：INFORMA HEALTHCARE  

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記述言語：日本語   掲載種別：速報，短報，研究ノート等（学術雑誌）   出版者・発行元：メディカルレビュー社  

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記述言語：日本語   出版者・発行元：(NPO)日本歯周病学会  

DOI： 10.14833/amjsp.2011f.0.93.0

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：W B SAUNDERS CO-ELSEVIER INC  

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記述言語：日本語   出版者・発行元：(一社)日本形成外科学会  

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記述言語：日本語   出版者・発行元：(NPO)日本歯周病学会  

DOI： 10.14833/amjsp.2009f.0.88.0

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記述言語：日本語   出版者・発行元：新潟歯学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2009113194

記述言語：日本語   出版者・発行元：新潟歯学会  

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記述言語：英語   出版者・発行元：AMER PHYSIOLOGICAL SOC  

Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP8-37. Am J Physiol Cell Physiol 289:C811-C818, 2005. First published June 15, 2005; doi:10.1152/ajpcell.00504.2004. Calcitonin gene-related peptide ( CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR(1)). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR(2), [Cys( Acm) 2,7] CGRP, and a relatively specific antagonist of CGRPR(1), CGRP8-37. [Cys( Acm) 2,7] CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein ( CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP8-37 potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA(2) value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino] ethyl)5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [I-125] CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR(1), and the second may be a novel variant of CGRPR(2).

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記述言語：英語   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: The biological actions of platelet-rich plasma (PRP) are thought to be mediated primarily by constituent transforming-growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-AB (PDGF-AB). However, we previously demonstrated that type I Collagen expression in periodontal ligament (PDL) cells is acutely stimulated through fibrin clot formation produced by the fibrinogen within PRP, rather than by the known growth factors. To investigate the possible involvement of other unidentified components in PRP action, we have now compared the effects of PRP with those of known recombinant growth factors on cell proliferation, alkaline phosphatase (ALP) activity, and collagen synthesis in human PDL cell cultures.
Methods: PRP was prepared by an established two-step centrifugation protocol using blood obtained from adult human volunteers. Cells cultured in serum-reduced medium on native or collagen-coated plates were treated with PRP, TGF-beta 1, or PDGF-AB. Cellular DNA synthesis was evaluated by bromodeoxyuridine incorporation. ALP activity was assessed using p-nitrophenyl phosphate with formalin-fixed cells, and cellular DNA content was subsequently quantified using bis-benzimide. Collagen synthesis was evaluated using a specific dye-based assay kit.
Results: 1) As did both TGF-beta 1 and PDGF-AB, PRP stimulated cell proliferation. 2) However, only the initial mitogenic action of PRP was attenuated in collagen-coated plates. 3) PRP, but neither growth factor, immediately induced fibrin clot formation and subsequently stimulated cellular adhesion and Collagen synthesis. 4) These effects were significantly augmented on collagen-coated plates. 5) PRP enhanced ALP activity, but neither TGF-beta 1 nor PDGF-AB replicated this effect.
Conclusions: When evaluated versus the concentrations of growth factors known to be contained by our PRP preparations, these data support the concept that PRP-constituent TGF-beta 1 acts as a significant growth factor on PDL cells. However, our findings also strongly suggest that the PRP-induced increase in ALP activity is mediated by an as-yet-unidentified component(s). In conjunction with previously demonstrated fibrinogen-mediated actions, our data provide evidence that PRP produces a number of potent effects on PDL cells that does not solely reflect simple combination of its major known growth factors.

DOI： 10.1902/jop.2005.76.5.760

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記述言語：日本語   出版者・発行元：日本口腔科学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2005188797

記述言語：日本語  

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記述言語：英語   出版者・発行元：AMER PHYSIOLOGICAL SOC  

Published data suggest that the neuropeptide calcitonin gene-related peptide ( CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca2+ concentration ([Ca2+](int)) and membrane potential (E-m) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)- trimethine oxanol [DiBAC(4)(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca2+] (int): a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC50 of similar to0.30 nM, and the maximal effect (initially similar to3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca2+ mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca2+ influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3', 5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca2+ discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca2+ influx through L-type voltage-dependent Ca2+ channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular Em, with maximal effect at 20 nM and an EC50 of 0.30 nM. This effect was attenuated with charybdotoxin ( - 20%) or glyburide ( glibenclamide; - 80%), suggesting that Em hyperpolarization is induced by both Ca2+-activated and ATP-sensitive K+ channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells.

DOI： 10.1152/ajpcell.00274.2003

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記述言語：英語  

In a series of studies, we have reported that 1,25-dihydroxyvitamin D 3, a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D3 and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D3 decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1α, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D3, but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D3. Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D3 on proliferating cells.

DOI： 10.1055/s-2004-825728

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

DOI： 10.1016/j.ejphar.2003.11.054

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：歯科基礎医学会  

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記述言語：英語   出版者・発行元：Elsevier  

Although accumulated data suggest that calcitonin gene-related peptide (CGRP) produces anabolic effects in skeletal tissue by directly acting on osteogenic cells, neither the distribution of CGRP receptor subtypes nor the associated cellular signaling pathways are well understood. In this study, we have pharmacologically and biochemically characterized CGRP-binding sites in immature human osteoblastic MG63 cells. In a [125I]CGRP whole-cell-binding assay, nonlinear regression curve-fitting analysis demonstrated a single binding site (KD=405±29 pM
13,100±223 sites per cell). Immunocytochemical and Western blot analyses demonstrated that 48-, 52-, and 120-kDa forms of the calcitonin receptor-like receptor (CRLR) and a 15-kDa form of the receptor-activity-modifying protein-1 (RAMP-1) was expressed on the plasma membrane. CGRP strongly stimulated cellular cAMP production and this effect was antagonized not only by an antagonist of the subtype-1 CGRP (CGRP1) receptor, CGRP-(8-37), but by an agonist of the putative subtype-2 CGRP (CGRP2) receptor, [Cys(Acm)2,7]-CGRP, that also itself acted as a weak agonist. In contrast to published data, CGRP dose- and time-dependently dephosphorylated and inactivated extracellular signal response kinase (ERK). This action was blocked by CGRP-(8-37), by an inhibitor of cAMP-dependent protein kinase (H-89), or by an inhibitor of protein phosphatases (vanadate). Prolonged CGRP treatments significantly suppressed DNA synthesis at 27 h, but up-regulated type I collagen. Both these actions were blocked by CGRP-(8-37) and mimicked by a specific inhibitor of ERK (PD98059). In summary, our data suggest that the CGRP receptors in MG63 cells meet many, but not all, of the classical criteria used to define CGRP1 receptors. These receptors that functioned in a pharmacologically distinct manner could inhibit cell proliferation, and were substantially more sensitive to a CGRP2 receptor agonist than are typical CGRP1 receptors. These receptor proteins were not exactly matched with the known components of a CGRP1 receptor that have been reported. Therefore, it is possible that the CGRP receptors expressed in immature osteoblastic human MG63 cells represent a variation of the known CGRP1 receptor.

DOI： 10.1016/S0014-2999(03)01763-1

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記述言語：英語   出版者・発行元：AMER ACAD PERIODONTOLOGY  

Background: Platelet-rich plasma (PRP) contains several growth factors, including platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), at high levels. We have demonstrated the PRP functions like TGF-beta to modulate cell proliferation in a cell-type specific manner. In addition, PRP forms gel-like materials in several, but not all, cell cultures tested. This study was designed to investigate PRP's action on extracellular matrix production in periodontal ligament (PDL) and osteoblastic MG63 cell cultures.
Methods: PRP was prepared from the plasma obtained from autologous blood of healthy volunteers and stored at -20degreesC until used. Cells treated with PRP (0.5% to 2%) were immunocytochemically stained for type I collagen and fibrin and the viscosity of the culture media was visually evaluated. Fibrinogen in PRP was detected by immunodot-blotting while endogenous thrombin expression in cells was detected by a modified enzyme-linked immunosorbent assay.
Results: Gel-like material rapidly (&lt;30 minutes) formed in cultures of either PDL or osteoblastic MG63 cell cultures after addition of PRP (greater than or equal to0.5%). PRP changed cell shape and up-regulated type I collagen at 24 hours. Fibrinogen was detected in the PRP preparations and insoluble fibrin networks were found in the newly formed gel-like material. PRP's action on collagen synthesis was mimicked by purified fibrinogen and blocked by thrombin inhibitor. Thrombin was expressed both in PDL and MG63 cells.
Conclusions: These findings demonstrated that the gel-like material formed in cell cultures of either PDL or MG63 cells is fibrin clot that is capable of up-regulating collagen synthesis in the extracellular matrix. Our data suggest the possibility that fibrinogen, converted to fibrin, in combination with growth factors present in PRP might effectively promote wound healing at sites of injury in periodontal tissue.

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記述言語：日本語   出版者・発行元：特定非営利活動法人日本歯周病学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC BONE & MINERAL RES  

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記述言語：英語   出版者・発行元：MUNKSGAARD INT PUBL LTD  

In our previous study, we demonstrated that porcine enamel matrix derivative (EMD) induces p21(WAF1/cip1) within 8 hours and subsequently arrests the cell cycle of human oral epithelial cells in G1 phase. In contrast, EMD markedly stimulates the proliferation of gingival fibroblasts without inducing p21(WAF1/cip1). To investigate the mechanism of how EMD produces these differential effects, we have focused on the initial response of these two cell types to EMD. In epithelial cell cultures, EMD stimulated cytoskeletal actin polymerization within 30 min and promoted cell adhesion within 3 hours, but EMD had no substantial effects on fibroblastic cell adhesion in our experimental system. EMD failed to stimulate either intracellular Ca2+ mobilization or CAMP production in either cell type. In both epithelial and fibroblastic cells, EMD (25-100 mug/ml) rapidly produced dose-dependent phosphorylation of the mitogen-activated protein kinase (MAPK) family: extracellular signal response kinase (ERK), p38-MAPK (p38-K), and c-Jun-terminal kinase/stress-activated protein kinase (JNK). However, neither inhibitors of MEK (ERK kinase) nor p38-K could block EMD's anti-proliferative action on epithelial cells. On the other hand, EMD rapidly stimulated translocation of smad2 into the nucleus in both cell types. Spurred by this finding, we assayed for TGF-beta1, a ligand for one receptor associated with smad2 activation, and detected significant levels in EMD preparations. The sutra of these pharmacological findings indicates that EMD contains at least one bioactive factor, which is most probably TGF-beta1 (or TGF-beta -like substances). In conjunction with the similarities in the differential growth-modulating actions between EMD and what is known for TGF-beta, we suggest that TGF-beta might act as the principal growth regulating agent of oral fibroblastic and epithelial cell types in EMD despite being present in only low levels.

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記述言語：日本語   出版者・発行元：特定非営利活動法人日本歯周病学会  

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記述言語：日本語   出版者・発行元：歯科基礎医学会  

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記述言語：英語   出版者・発行元：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

The effects of N-G-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase (NOS) inhibitor, on the L-type Ca2+ current (ICa) and NO effects on NOS were determined in rat ventricular myocytes. L-NMMA (10 and 100 mu M) had no significant effect on basal ICa, but in a cAMP-stimulated condition due to forskolin (1 mu M) or milrinone (10 mu M), a cGMP-inhibited cAMP-phosphodiesterase (PDE), L-NMMA (10 and 100 mu M) concentration dependently augmented ICa. The enhancing effects of L-NMMA (10 and 100 mu M) on ICa were not seen in the presence of either a nonselective inhibitor of PDE, 3-isobutyl-1-methylxanthine (20 mu M), resulting in a stimulated ICa condition or a cGMP-dependent protein kinase activator, 8-bromo-cGMP (200 mu M). 8-Bromo-cGMP (200 mu M) inhibited 100 mu M L-NMMA-induced ICa increase in the simultaneous application of forskolin (1 mu M). Acetylcholine (ACh; 1 and 3 mu M) inhibited 1 mu M forskolin-stimulated ICa in a concentration-dependent manner, but this inhibitory action of ACh was significantly attenuated by the additional application of L-NMMA (100 mu M). In the continuing presence of both L-NMMA (100 mu M) and forskolin (1 mu M), ACh (6 mu M) had no inhibitory effect on ICa. In another series of experiments with isolated ventricular myocytes, we obtained both the positive staining of NADPH-diaphorase activity and the expression of the endothelial isoform of NOS. These data suggest that the effect of L-NMMA on ICa in a cAMP-stimulated condition with or without cholinergic inhibition is due to inhibition (acute effects) of a cGMP-stimulated cAMP-PDE via inhibition of the endothelial isoform of NOS.

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記述言語：日本語   出版者・発行元：特定非営利活動法人日本歯周病学会  

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記述言語：日本語   出版者・発行元：特定非営利活動法人日本歯周病学会  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：AMER SOC BONE & MINERAL RES  

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記述言語：日本語   出版者・発行元：歯科基礎医学会  

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記述言語：日本語   出版者・発行元：特定非営利活動法人日本歯周病学会  

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記述言語：英語   出版者・発行元：SPRINGER VERLAG  

In previous studies we found that the calciotropic hormone 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] augments the action of either prostaglandin E-1 (PGE(1)) or NaF to induce differentiation of human promyelocytic HL-60 cells, a process that features increased generation of nitric oxide (NO) via up-regulation of inducible nitric oxide synthase (iNOS). We have now examined the short-term interaction of 1,25(OH)(2)D-3 with phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) in these cells. PMA (100 nM) alone generally up-regulated several classical indices of macrophagic differentiation and stimulated cellular production of interleukin (IL)-1 alpha, IL-6, tumor-necrosis factor (TNF)-alpha, PGE(2), and NO. Increased generation of NO primarily resulted from increased expression of cellular iNOS. When 1,25(OH)(2)D-3 (10 nM) was added to PMA treatments, most PMA-induced changes, particularly its effects to up-regulate iNOS-dependent NO production and change cell morphology, were multiplicatively augmented. In contrast, DMSO (1.3%) alone, an inducer of granulocytic differentiation, increased cytokine production, but failed to stimulate NO production or induce iNOS. In contrast to its striking interaction with PMA, 1,25(OH)(2)D-3 could not augment DMSO's differentiative effects. Changes in cellular cytokine production were eliminated as the driving force in HL-60 differentiation when specific neutralizing antibodies failed to produce any attenuation of iNOS up-regulation or of the shifts in cell morphology. However, indomethacin (30 mu M) blocked the synergistic interaction between 1,25(OH)(2)D-3 + PMA to shift cell morphology and stimulate NO production. Subsequently adding PGE(2) (1 ng/ml) to indomethacin-treated cells restored the ability of 1,25(OH)(2)D-3 + PMA to interactively increase cellular NO production, but failed to fully replicate the strong shift in cell morphology typical of PMA + 1,25(OH)(2)D-3 treatments. Our findings suggest that interaction between 1,25(OH)(2)D-3 and PMA to induce macrophagic differentiation increases iNOS-dependent NO production by a mechanism involving a cyclooxygenase product(s), possibly PGE(2).

DOI： 10.1007/s002239900485

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL SCIENCE INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL SCIENCE INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL SCIENCE INC  

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記述言語：英語   出版者・発行元：ENDOCRINE SOC  

In certain neurons, alternative RNA processing generates calcitonin gene-related peptide (CGRP) from the same gene that encodes the hormone calcitonin. As CGRP-containing nerve fibers are prominent in skeleton, we evaluated the effects of CGRP on osteoblasts. Because the vasodilatory effect of neural CGRP in smooth muscle probably involves inhibition of unstimulated Ca2+ uptake, we examined the acute effects of CGRP on this parameter in rat osteoblastic cells. CGRP inhibits Ca-45(2+) uptake in both UMR 106 osteosarcoma and RCOB-3 osteoblastic cells. This inhibition is rapid (0.5 min), occurs with an EC(50) of 1 nM, and cannot be demonstrated in the presence of 0.1 mM diltiazem, a blocker of voltage-dependent Ca2+ channels. Depolarization of bone cells with high extracellular potassium (K+) also blocks the effect of CGRP on Ca-45(2+) uptake, suggesting a central role for K+ channels in mediating this action. In agreement with this hypothesis, the effect of CGRP is blocked by 1 mu M glybenclamide, a specific inhibitor of ATP-sensitive potassium (K-ATP) channels, or by pretreatment of cells with 1 mM iodoacetic acid to deplete intracellular ATP, Blocking Ca2+-activated potassium channels with 1 mM tetraethylammonium does not prevent CGRP's effect. Pinacidil, a specific activator of K-ATP channels, mimics CGRP's effect. Both CGRP and pinacidil also produce a small significant stimulation of cellular Ca2+ efflux in UMR 106 cells. These data suggest that inhibition of diltiazem-sensitive Ca2+ channels occurs secondary to the hyperpolarization engendered by CGRP activation of K-ATP channels in osteoblastic cells, an effect similar to that of CGRP on smooth muscle cells.

DOI： 10.1210/en.137.3.984

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL SCIENCE PUBL INC CAMBRIDGE  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL SCIENCE INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL SCIENCE INC  

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：BLACKWELL SCIENCE INC  

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記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

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記述言語：日本語   出版者・発行元：新潟歯学会  

我々は,マウス新生仔頭蓋骨より分離した骨細胞をアルカリホスファターゼ(ALP)活性を維持した状態で6カ月以上35代まで継代培養できたことについて報告する｡造骨細胞は, マウス新生仔頭蓋骨よりcoll-genase / dispase II 処理によって分離し,α-MEM+15%FCSで5%CO_2, 37℃ 条件下で培養した｡継代数3-6代で培加時間が最も長くなったが,それ以降は増殖率も高まると同時に形態的にも安定した｡この細胞のALP活性は,コンフルエントシート形成後,経時的に上昇する傾向を持ち,それに伴い石灰化物の形成が観察された｡また,insulin, prostaglandin E_2(PGE_2),血清に応答してALP活性の上昇が観察された｡以上の結果から,我々の維持している細胞は骨芽細胞様であることが示唆された｡

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その他リンク： http://search.jamas.or.jp/link/ui/1988122285

記述言語：日本語   出版者・発行元：新潟歯学会  

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その他リンク： http://search.jamas.or.jp/link/ui/1988020390